Specific IgE levels overlapped between allergic and tolerant donors
In order to study differences in antibody repertoires between peanut allergic and peanut sensitized (IgE) but tolerant patients, blood was drawn from peanut allergic (n=6, age: 27-41) and tolerant (n=6, age: 27-63) donors sensitized to Ara h2 and/or 6 (≥0.1 kU/l). Specific IgE levels to Ara h2 ranged from 1.0- 72 kU/l in the allergic and from 0-1.7 kU/l in the tolerant group. Specific IgE levels to Ara h 6 were lower compared with sIgE levels to Ara h2 and ranged from 0-13 kU/l in the allergic and from 0-0.9 kU/l in the tolerant group. Mono-sensitization to either Ara h2 or 6 was detected in both groups, although it occurred more often within the tolerant group (allergic: Pt-01, tolerant: Pt-02, 03, 07) as shown in Table 1.
Frequency of peanut 2S albumin-binding B-cells was significantly higher in allergic donors
As a first step, the frequencies of 2S albumin-binding B-cells were compared between peanut allergic patients, peanut sensitized but tolerant patients, and non-atopic controls. 2S albumin-binding B-cells, double-positive for allergen-tetramer staining, were defined as putatively specific, and their frequency was expressed as percentage of total B-cells acquired from the respective sample. While the frequency in allergic patients (median: 0.01%, 95% CI: 0.005-0.164) was only slightly increased compared to the tolerant patients (median: 0.006%, 95% CI: 0.0016-0.014), the frequency was significantly elevated compared with non-atopic controls (median: 0.002%, 95% CI: 0.0004-0.004, p=.008). Those 2S albumin-binding B-cells were single-cell sorted and served as source for heavy and corresponding light chain gene transcript amplification. No correlation was found between the frequency of 2S albumin-binding B-cells and the number of 2S albumin-binding B-cells from which the heavy and the corresponding light chain gene transcripts were successfully amplified and sequenced. A high number of V(D)J gene transcripts were successfully amplified from the B-cells of patients 4 and 6 – both allergic – while none or only one V(D)J gene transcript was successfully amplified from B-cells of patient 2 (tolerant) and 9 (allergic), respectively. For both of these patients, only a small number of 2S albumin-binding B-cells were sorted (Pt-2: 8, Pt-9: 12), which may exclude amplification failure as a reason for obtaining such a low number of successfully amplified gene transcripts. Frequencies, number of sorted 2S albumin-binding B-cells and successfully amplified corresponding V(D)J gene transcripts are shown in Figure 111Sorted B-cells/amplified corresponding V(D)J gene transcripts - allergic: Pt-1 (32/49), Pt-4 (63/192), Pt-5 (8/96), Pt-6 (61/192), Pt-9 (1/12), Pt-10 (19/96); tolerant: Pt-2 (0/8), Pt-3 (7/108), Pt-7 (13/40), Pt-8 (7/36), Pt-11 (36/144), Pt-12 (17/72); non-atopic: NA-1 (21/50), NA-2 (14/32), NA-3 (7/36), NA-4 (8/48), NA-5 (12/24). Taken together, those successfully sequenced gene transcripts - heavy chain: 280, light chain: 221 - provide a good basis for further analyses.