Single B-cell deconvolution of peanut-specific antibody responses in allergic patients

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• 作者：Ramona A. Hoh ; PhD^a ; Shilpa A. Joshi ; PhD^a ; Yi Liu ; PhD^a ; ^b ; Chen Wang ; PhD^a ; Krishna M. Roskin ; PhD^a ; Ji-Yeun Lee ; BS^a ; Tho Pham ; MD^a ; Tim J. Looney ; PhD^a ; Katherine J.L. Jackson ; PhD^a ; Vaishali P. Dixit ; BA^a ; Jasmine King ; BS^a ; Shu-Chen Lyu ; MS^c ; Jennifer Jenks ; BS^c ; Robert G. Hamilton ; PhD^d ; Kari C. Nadeau ; MD ; PhD^c ; Scott D. Boyd ; MD ; PhD^a ; ^{sboyd1@stanford.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Peanut ; allergy ; B cell ; antibody ; IgE ; IgG4 ; immunotherapy ; high-throughput DNA sequencing ; antigen specific ; allergen specific ; repertoire ; somatic mutation
• 刊名：Journal of Allergy and Clinical Immunology
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：137
• 期：1
• 页码：157-167
• 全文大小：2716 K

文摘

The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance.

d="absSec_2">Objective

d="abspara0015">We sought to characterize peanut allergen–specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy.

d="absSec_3">Methods

d="abspara0020">B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones.

d="absSec_4">Results

d="abspara0025">Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG₄ members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase.

d="absSec_5">Conclusion

d="abspara0030">Most peanut allergen–binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG₄.