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α-gal特异性人IgE抗体促进α-gal诱导及抗原非依赖性过敏效应细胞活化
Alpha-gal-specific human IgE promotes both alpha-gal-induced and antigen-independent allergic effector cell activation
摘要
Background: In alpha-gal syndrome (AGS), immunoglobulin (Ig)E antibodies form against the glycan galactose-alpha-1,3-galactose (alpha-gal) in mammal products rather than food proteins. Alpha-gal glycolipids and glycoproteins activate human basophils sensitized with AGS plasma in an IgE-dependent fashion. However, it is unknown whether alpha-gal-specific IgE, independent of other blood proteins, is sufficient for mediating basophil and mast cell activation.
Objective: We determined whether alpha-gal antigens could activate passively-sensitized rat basophil leukemia (RBL) SX-38 cells, which express human IgE receptors and are commonly used to model allergen / IgE-mediated mast cell activation in food-protein allergy.
Methods: Using CRISPR, we created a novel, alpha-gal-deficient RBL cell line, AGKO RBL SX-38, passively sensitizing cells with sera from AGS donors or with novel alpha-gal-specific IgE clones, then stimulating with alpha-gal glycoproteins. To assess effector cell activation, we measured cell surface expression of activation marker CD63 by flow cytometry and mediator release through β-hexosaminidase release assays.
Results: After alpha-gal-antigen stimulation, percentages of CD63+ AGKO RBL SX-38 cells sensitized with AGS sera increased 3-fold compared to cells sensitized with control serum. Select human AGS IgE clones facilitated alpha-gal-antigen-dependent and antigen-independent CD63 upregulation. Cells sensitized with pooled AGS sera released β-hexosaminidase in an alpha-gal-independent fashion. We saw no β-hexosaminidase release above background in cells sensitized with alpha-gal-specific IgE clones.
Conclusion: Certain alpha-gal-specific human IgE clones may partially activate allergic effector cells independent of antigen, potentially lowering thresholds for subsequent alpha-gal-induced or antigen-independent allergic effector cell degranulation. This may affect duration and severity of allergic symptoms in patients with AGS.
实验方法
产品清单
| 名称 | 品牌 | 货号 |
|---|---|---|
| 最低必需培养基 | Gibco | -- |
| 胎牛血清 | Corning | -- |
| G418（遗传霉素） | Corning | -- |
| StemPro-34培养基 | -- | -- |
| 干细胞因子 | GIBCO | -- |
| 核转染试剂盒 | Lonza | V4XC-2032, SF cell line 4D-Nucleofector kit |
| 流式细胞分选仪 | Becton Dickinson | -- |
| 96孔板 | -- | -- |
| 裂解缓冲液 | Cell Signaling Technology | 9803S |
| BCA蛋白测定试剂盒 | Thermo Scientific | 23225 |
| 4%-20% SDS-PAGE凝胶 | Invitrogen | -- |
| 硝酸纤维素膜 | -- | -- |
| 抗α-gal单克隆抗体 | Enzo Life Sciences | -- |
| 山羊抗小鼠IgM HRP偶联二抗 | -- | -- |
| 动态光散射板式检测仪 | Wyatt | DynaPro PlateReader-II |
| 384孔微孔板 | -- | -- |
| 牛甲状腺球蛋白 | Sigma | T100-500MG |
| α-gal-人血清白蛋白 | Dextra | NGP3203 |
| 西妥昔单抗 | Eli Lilly | -- |
| DPBS-牛血清白蛋白 | Merck | 126575-100MG |
| HRP偶联抗人IgE Fc抗体 | Southern Biotech | 9160-05 |
| Hank's平衡盐溶液缓冲液 | Gibco | -- |
| 内毒素去除柱 | Pierce | -- |
| Triton X-100 | -- | -- |
| 细胞刮刀 | -- | -- |
| 5毫升圆底聚苯乙烯流式管 | -- | -- |
| Zombie-red活死细胞染色剂 | Biolegend | 423109 |
| BV421标记抗人FcεRI抗体 | Biolegend | AER-37 |
| AF647标记抗人IgE抗体 | Biolegend | MHE-18 |
| FITC标记抗大鼠CD63抗体 | Miltenyi Biotec | REA444 |
| 流式细胞分析仪 | BD Biosciences | -- |
| 24孔板 | -- | -- |