Intravenous immunoglobulin induces a functional silencing program similar to anergy in human B cells  Jean-François Séïté, PhD, Carole Goutsmedt, MSc, Pierre Youinou, MD, PhD, Jacques-Olivier Pers, DDS, PhD, Sophie Hillion, PhD  Journal of Allergy and Clinical Immunology  Volume 133, Issue 1, Pages 181-188.e9 (January 2014) DOI: 10.1016/j.jaci.2013.08.042 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IVIg suppresses BCR-mediated activation. B cells were stimulated with anti-IgM in the presence or absence of IVIg, SA-IVIg, or BSA. A, CD40, CD80, and CD86 expression levels on 20-hour stimulated cells were determined by using FACS. Data are presented as means ± SEMs of 5 independent experiments. ***P < .001. B, Efficiency of IVIg, F(ab')2-IVIg, or Fc-IVIg in inhibition of B-cell activation. Tonsillar B cells were stimulated for 24 hours with anti-IgM, and inhibitory effects of 20 mg/mL IVIg, 10 mg/mL F(ab′)2-IVIg, or 10 mg/mL Fc-IVIg were evaluated based on CD80 expression measurement. Data (means ± SEMs) are from 4 independent assays. *P < .05 and **P < .01. C, Lysates from 10-minute stimulated B cells were immunoblotted with anti–p-Tyr and anti–β-actin mAbs. D and E, B cells were loaded with Fluo-4 AM and BCR stimulated (Fig 1, D) or incubated with IVIg for 2 hours, washed, and cultured overnight before BCR stimulation (Fig 1, E). Ionomycin was used as a positive control. Data are representative of 5 independent experiments. NS, Not significant. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IVIg disrupts BCR aggregation in lipid rafts. A and B, B cells were incubated at 4°C (Fig 2, A) or 37°C (Fig 2, B) with biotin-conjugated anti-human IgM antibody, TRITC-conjugated avidin and FITC-conjugated IVIg (Fig 2, A), or FITC-labeled CTB in the presence or absence of IVIg (Fig 2, B). Colocalization of IgM and IVIg was evaluated by using the Pearson correlation coefficient. Data (means ± SEMs) are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 IVIg increases BCR internalization. B cells were cultured with anti-IgM antibody with or without IVIg (or SA-IVIg or BSA). A, The remaining surface expression of BCRs was determined by using FACS with FITC-conjugated anti-goat IgG antibody. **P < .01 and ***P < .001. ns, Not significant. B, BCR cross-linking was achieved with biotin-conjugated anti-IgM and revealed with TRITC-conjugated streptavidin. CD22 was stained with FITC-conjugated anti-CD22. Data are representative of 3 independent experiments. C, B cells were stimulated for 5 minutes, and cell lysates were immunoblotted with anti-phosphorylated CD22 (pCD22), anti–pan-CD22, anti-phosphorylated CD32 (pCD32b), or anti–pan-CD32 antibodies. Data are representative of 5 experiments. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 PI3K/Akt signaling pathway is suppressed by IVIg. B cells were stimulated with anti-IgM antibody for 5 minutes (Fig 4, A, B, and D) or 16 hours (Fig 4, C and D) with or without IVIg (or SA-IVIg). A and B, Production of PIP3 (Fig 4, A) and phosphorylation of CD19 (Y513; Fig 4, B) were studied by using FACS after a 5-minute stimulation. C and D, Cell lysates were immunoblotted with anti-phosphorylated Akt (pAkt) and anti–pan-Akt antibodies (Fig 4, C) or with anti–SHP-1, anti–SHIP-1, anti-PTEN, and anti–β-actin mAbs (Fig 4, D). Data are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 IVIg has distinct effects on NF-κB and NFAT signaling pathways. B cells were cultured for 1, 3, and 6 hours with anti-IgM antibody with or without IVIg. Nuclear proteins were fractionated, and lysates were immunoblotted with anti-p65, anti–c-Rel, anti-NFAT2, or anti–histone H3 mAbs. Data are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Transcriptional activity of B cells is modified by IVIg. Quantitative PCR analyses of changes in the expression of 80 transcription factors were performed in B cells untreated (control group) or stimulated for 90 minutes with anti-IgM antibody with or without IVIg. Results are presented as fold changes in gene expression in the tested groups relative to the control group. CEBPB, CCAAT/enhancer-binding protein beta; CREBBP, CREB-binding protein; Ct, cycle threshold; EGR1, early growth response protein 1; FOS, FBJ murine osteosarcoma viral oncogene homolog; ID1, DNA-binding protein inhibitor; IRF1, interferon regulatory factor 1; MYC, v-myc avian myelocytomatosis viral oncogene homolog; NFKB1, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; POU2AF1, POU class 2 associating factor 1; RB1, retinoblastoma 1; SP1, Sp1 transcription factor; TP53, tumor protein p53. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Comparison of F(ab')2 anti-IgM versus anti-IgM whole IgG efficiency in activation of B cells in the presence or absence of IVIg. Tonsillar B cells were stimulated for 24 hours with 10 or 20 μg/mL F(ab')2 or whole IgG anti-IgM antibodies. Inhibitory effect of IVIg on B-cell activation was evaluated on induction of CD80 and CD86 expression. Data (means ± SEMs) are from 6 independent assays. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Effects of free Neu5Ac and synthetic Neu5Acα2-6Gal-PAA on BCR-mediated activation. A, Binding of biotin-conjugated Neu5Acα2-6Gal-PAA on B cells in the absence (dots) or presence (squares) of IVIg was evaluated by means of flow cytometry. B, Colocalization of biotin-conjugated Neu5Acα2-6Gal-PAA and CD22 on B cells was assessed by using confocal microscopy. C and D, Long-term (16 hours) and short-term (4 minutes) inhibitory effects of free Neu5Ac and Neu5Acα2-6Gal-PAA on BCR-mediated activation were assessed by means of evaluation of CD80 expression by using flow cytometry (Fig E2, C) and by using determination of phosphorylation of CD22, Syk, Erk, or Tyr by Western blotting (Fig E2, D), respectively. Data are representative of 3 independent assays (Fig E2, A, B, and D) or obtained from 3 independent assays (Fig E2, C; mean ± SEM). Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Genes differentially expressed with a 2-fold change between the tested conditions and control conditions are presented on Venn diagrams. Upregulated genes are shown in the left panel, and downregulated genes are shown in the right panel. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Clustergram of transcription factors. Coregulated genes across samples are displayed on a heat map to visualize clusters. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Confirmation of RT2 PCR array: egr1, nfkb1, p53, sp1, and id1 gen expressions are modulated by IVIg. Expression levels of egr1, nfkb1, p53, sp1, and id1 were studied by using quantitative PCR on cDNA obtained from B cells after a 90-minute stimulation with anti-IgM antibody in the presence or absence of IVIg. Expression levels were normalized to 18S, which was used as an internal control, before calculation of fold change and compared with those in unstimulated cells. Five independent experiments are represented. P values were calculated by using the paired t test as follows: **P < .01 and *P < .05. Journal of Allergy and Clinical Immunology 2014 133, 181-188.e9DOI: (10.1016/j.jaci.2013.08.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions