Discussion
Specific IgE levels to Ara h2 and 6 between 0.1 and 1.8 kU/l overlap between peanut allergic and tolerant subjects5, 6, risking inaccurate diagnosis, and therefore indicating the need for new diagnostic strategies. In the present study, we observed a preferential usage of VH3-family genes in peanut 2S albumin-specific B-cells from peanut allergic patients. Additionally, we identified 2S albumin-binding B-cells carrying HCDR3 sequence motifs either related to peanut allergy or tolerance. This finding may lead towards new diagnostic strategies able to discriminate between allergy and tolerance in sensitized patients with suspected peanut allergy.
Despite a large number of amplified heavy chain VDJ gene transcripts (n=280), only three of them belonged to IgE class-switched B-cells. This low abundance may be potentially explained by dominant class-switching to IgE in the tissue19, low BCR surface expression of plasmablast-like IgE+ B-cells28-30 and the extreme low abundance of IgE+ B-cells within the circulation23. Nevertheless, the relevance of the B-cells sorted within the present study is supported by the close relationship between one heavy chain gene transcript from an IgE+ B-cell and a gene transcript from an IgG1+ B-cell derived from an unrelated peanut allergic donor. Additionally, some generated mAbs were able to slightly inhibit patient-derived serum IgE-binding to Ara h2 and 6 (Supplementary, Figure S3). Correspondingly, there is evidence that human IgE+ B-cells are predominately plasmablasts or plasma cells generated by sequential class-switching from IgG1+ B-cells (γ switch region remnants) and that the humoral IgE memory is contained in IgG+ memory B-cells21. Further evidence for this theory was obtained by clonal relationship analyses, since IgE+ B-cells were dominantly clonally related to IgG1+ B-cells, but also, to a lesser extent, to B-cells of other isotypes19, 20, 28.
By contrast to the low number of IgE+ B-cells, IgM+ B-cells represented the largest isotype group, as detected in respectively 47, 53 and 94% of allergic, tolerant and non-atopic donors. Additionally, IgM+ B-cells from non-atopic donors, all specific for Ara h2 and/or 6, shared HCDR3 motifs (motif 4, 8, 10) with IgM+, IgG2+ and IgA1+ B-cells from allergic donors. Clonal relationships between IgA/G/M+ and IgE+ B-cells have been described for B-cells derived from gut tissues by Hoh and colleagues19, 31. Combining the findings from the present study with the finding of Hoh and colleagues leads to the suggestion that non-atopic donors can potentially possess non-IgE antibodies with required specificity or affinity to theoretically induce an allergic reaction.
Moreover, tolerant donors showed a higher proportion of IgA+ B-cells and tended to have higher specific IgA serum levels than allergic donors (Supplementary, Figure S3). Allergen-specific IgA serum levels have been shown to be increased in peanut allergic subjects undergoing oral or sublingual immunotherapy compared with their baseline levels. Moreover, salivary IgA levels have been closely associated with the degree of tolerance induction confirmed by DBPCFCs, pointing to a protective role of specific IgA against mucosal allergen absorption22, 32. Such a protective role is supported by increased intestinal permeability in the absence of IgA in mice33. Taken together, these findings suggest a potential protective effect of allergen-specific IgA in the mucosa of sensitized but tolerant patients.
Regarding gene lineage, VH3-family genes were significantly more often used (p < .0001) in heavy chain gene transcripts of peanut 2S albumin-specific B-cells from allergic donors (89%) than in those from tolerant (54%) and non-atopic donors (63%). Previous studies on VH-gene usage of heavy chain gene transcripts in healthy donors showed VH3-family gene usage in 40 to 65% of the B-cells, which was comparable to the usage in the tolerant and non-atopic groups in our study (Table 2)34, 35. These findings suggest a shift towards VH3-family gene usage in 2S albumin specific B-cells from allergic donors. Contrary to our findings, other datasets of Ara h2 specific B-cells did not observe a similar shift in VH-gene usage11, 14. These datasets, however, were generated from patients undergoing peanut oral immunotherapy with a focus on different time points during immunotherapy. This fact hampers the comparison between the present and previous Ara h2-related datasets. Moreover, conflicting results regarding preferred VH-gene usage were described for different food and respiratory allergies, with a shift to VH3 usage for anti-alpha Gal antibodies and anti-grass pollen Phl p6 and 11 antibodies36, 37, indicating that the preferred VH-family gene usage may be allergen dependent. Moreover, the preferred usage might depend on the status of allergy or tolerance and can potentially be used for diagnostic purposes upon validation in a larger patient cohort. A validation study will also provide information about the number of detected 2S albumin-binding B-cells is sufficient for a diagnostic workflow.
Besides differences in VH-family gene usage, certain HCDR3 sequence motifs were associated with either peanut allergy or tolerance and appear to have the ability to discriminate between those two groups. The main HCDR3 sequence motif ‘CARDSSALEIYNRFDPW’ was associated with peanut allergy and derived from recombined VH3-30, DH3-3 and JH5 genes. Comparably, VH3-30*18 was incorporated in the VDJ gene transcript of clonally related IgE+ B-cells specific for Ara h2 in the study of Croote and colleagues28. Additionally, a highly similar HCDR3 region (CAREGYESSGFDYW) to motif 6 (CAREGHY SSQ FDYW), associated with tolerance, has been described for peanut allergic subjects undergoing oral immunotherapy. Oral immunotherapy may shape the antibody repertoire towards repertoires present in tolerant subjects14. These comparisons support the potential of HCDR3 motifs in diagnosing peanut allergy and tolerance.
In conclusion, the dominant usage of VH3-family genes and the identification of HCDR3 sequence motifs related to either peanut allergy or tolerance may potentially lead to the development of new diagnostic strategies for subjects with suspected peanut allergy and sensitization to Ara h2 and/or 6. Validation of these HCDR3 sequence motifs in a larger patient cohort may be achieved using next-generated sequencing approaches, potentially combined with the sorting strategy of 2S albumin-binding B-cells presented in this study. Next-generation sequencing allows high-throughput and can be more easily implemented in diagnostic routine38, 39.