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. 2023 May;151(5):1357-1370.e9.
doi: 10.1016/j.jaci.2022.11.030. Epub 2023 Jan 14.

Single-cell RNA sequencing identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract

Janice A Layhadi  1 , Raquel Moya  2 , Tiak Ju Tan  1 , Madison M Lenormand  1 , Hanisah Sharif  3 , Rebecca V Parkin  1 , Gemma Vila-Nadal  1 , Oleksandra Fedina  1 , Rongfei Zhu  1 , Wannada Laisuan  1 , Stephen R Durham  1 , Jerónimo Carnés  2 , Mohamed H Shamji  4
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Free article

Single-cell RNA sequencing identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract

Janice A Layhadi et al. J Allergy Clin Immunol. 2023 May.
Free article

Abstract

Background: Immunologic mechanism of action of allergoids remains poorly understood. Previous models of allergenicity and immunogenicity have yielded suboptimal knowledge of these immunotherapeutic vaccine products. Novel single-cell RNA sequencing technology offers a bridge to this gap in knowledge.

Objective: We sought to identify the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized Phleum pratense (Phl p) extract.

Methods: The molecular mechanisms underlying native Phl p, depigmented Phl p (DPG-Phl p), and depigmented-polymerized (DPG-POL-Phl p) allergoid were investigated by single-cell RNA sequencing. Allergen-specific TH2A, T follicular helper (Tfh), and IL-10+ regulatory B cells were quantified by flow cytometry in peripheral blood mononuclear cells from 16 grass pollen-allergic and 8 nonatopic control subjects. The ability of Phl p, DPG-Phl p, and DPG-POL-Phl p to elicit FcεRI- and FcεRII-mediated IgE responses was measured by basophil activation test and IgE-facilitated allergen binding assay.

Results: Analysis revealed that DPG-POL-Phl p downregulated genes associated with TH2 signaling, induced functional regulatory T cells exhibiting immunosuppressive roles through CD52 and Siglec-10, modulated genes encoding immunoproteasome that dysregulate the processing and presentation of antigens to T cells and promoted a shift from IgE toward an IgA1 and IgG responses. In grass pollen-allergic subjects, DPG-POL-Phl p exhibited reduced capacity to elicit proliferation of TH2A, IL-4+ Tfh and IL-21+ Tfh cells while being the most prominent at inducing IL-10+CD19+CD5hi and IL-10+CD19+CD5hiCD38intCD24int regulatory B-cell subsets compared to Phl p (all P < .05). Furthermore, DPG-POL-Phl p demonstrated a hypoallergenic profile through basophil activation and histamine release compared to Phl p (31.54-fold, P < .001).

Conclusions: Single-cell RNA sequencing provides an in-depth resolution of the mechanisms underlying the tolerogenic profile of DPG-POL-Phl p.

Keywords: Depigmented-polymerized allergen; allergen immunotherapy; allergic rhinitis; allergoid; mechanism of action; single-cell RNA-Seq.

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