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.