Alpha-gal–specific human IgE promotes both alpha-gal–induced and antigen-independent allergic effector cell activation - 03/04/26

Doi : 10.1016/j.jaci.2025.12.1007

Yinghui Wang, MBBS, MS ^a,^b,^c,^d, Yugen Zhang, DVM, PhD ^a,^b,^d, Christa Dudley, BS ^a,^b,^d, Katherine Fitzgerald, BS ^a,^b,^d, Audrey S. Carson, BS ^a,^b,^d, Camille M. Kapita, MS ^a,^b,^d, Daaniya Rana ^a,^b,^d,^{^{∗
Daaniya Rana does not yet have a terminal degree.}}, Kim Kluckman, MBA ^e, Donna Bortner, PhD ^e, Dale O. Cowley, PhD ^e, Christopher R. Travis, PhD ^f, Julia Vorobiov, BA ^a,^b,^d, Shailesh Choudhary, PhD ^a,^b,^d, Christopher L. Kepley, PhD ^g, Scott P. Commins, MD, PhD ^a,^b,^d, Scott A. Smith, MD, PhD ^h,^i,^j, Onyinye I. Iweala, MD, PhD ^a,^b,^c,^d,^⁎

^a Division of Rheumatology, Allergy, and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC

^b Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC

^c Allergy Mast Cell Disorders Program, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

^d Division of Allergy and Immunology, Department of Pediatrics, Food Allergy Initiative, University of North Carolina at Chapel Hill, Chapel Hill, NC

^e UNC Animal Models Core, University of North Carolina at Chapel Hill, Chapel Hill, NC

^f UNC Macromolecular Interactions Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC

^g Liberty University College of Osteopathic Medicine, Lynchburg, Va

^h Department of Medicine, Vanderbilt University Medical Center, Nashville, Tenn

ⁱ Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tenn

^j Division of Asthma, Allergy, and Clinical Immunology, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Va

^∗ Corresponding author: Onyinye I. Iweala, MD, PhD, Division of Rheumatology, Allergy, and Immunology, University of North Carolina at Chapel Hill, 3300 Thurston Bldg, CB#7280, Chapel Hill, NC 27599. Division of Rheumatology, Allergy, and Immunology University of North Carolina at Chapel Hill 3300 Thurston Bldg CB#7280 Chapel Hill NC 27599

Graphical abstract

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Abstract

Background

In alpha-gal syndrome (AGS), IgE antibodies form against the glycan galactose-α-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 (sIgE), independent of other blood proteins, is sufficient for mediating basophil and mast cell activation.

Objective

We sought to determine whether alpha-gal antigens could activate passively sensitized rat basophilic 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 the clustered regularly interspaced short palindromic repeats technology, we created a novel, alpha-gal–deficient RBL cell line, alpha-gal knockout RBL SX-38, passively sensitizing cells with sera from AGS donors or with novel alpha-gal sIgE clones, and then stimulated 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, the percentage of CD63 ⁺ alpha-gal knockout RBL SX-38 cells sensitized with AGS sera increased 3-fold compared with 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 sIgE clones.

Conclusions

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.

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Key words : Alpha-gal, alpha-gal syndrome, galactose-α-1,3-galactose, mast cell, basophil, human IgE, RBL SX-38

Abbreviations used : alpha-gal–HSA, AGKO, AGS, alpha-gal, BAT, BTG, CRISPR, DPBS, FACS, LAD2, MEM, RBL SX-38, sIgE, UNC

Plan

Methods

Study approval

Cell culture

CRISPR-Cas9 to generate AGKO RBL SX-38 cells

Western blot

Dynamic light scattering experiments

ELISAs

β-Hexosaminidase release assay

Flow cytometry

Statistical analysis

Results

Creation of alpha-gal–deficient AGKO RBL SX-38 cells using CRISPR

Human AGS sera promote alpha-gal antigen-dependent and antigen-independent CD63 expression on RBL SX-38 cells

Select alpha-gal–specific human IgE clones induce both alpha-gal antigen-dependent and antigen-independent CD63 expression on RBL SX-38 cells

Molecular characterization of alpha-gal–specific human IgE clones show varying aggregation states

Alpha-gal–specific human IgE mAbs show differential recognition of different alpha-gal glycoproteins

AGS sera, but not alpha-gal–specific human IgE clones, induce β-hexosaminidase release from AGKO RBL SX-38 cells

Discussion

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Vol 157 - N° 4

P. 932-946 - avril 2026 Retour au numéro

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Alpha-gal–specific human IgE promotes both alpha-gal–induced and antigen-independent allergic effector cell activation - 03/04/26

Graphical abstract

Abstract

Background

Objective

Methods

Results

Conclusions

Plan

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