Allergic and tolerant donors possess more class-switched 2S albumin-binding B-cells than non-atopic controls
All successfully sequenced heavy chain gene transcripts were initially analyzed for isotype distribution, as class-switching is mostly accompanied by somatic hypermutation maturation upon antigen challenge. Overall, IgM was the most dominant isotype across all groups. Notably, its prevalence was much lower in allergic (47%) and tolerant (53%) donors than in the non-atopic reference group (94%), indicating more antigen-challenged and matured specific B-cells originated from allergic and tolerant patients. Moreover, the prevalence of IgA2-expressing B-cells was increased in the tolerant group (20%), while a comparable distribution was observed in allergic and non-atopic donors (2%), as shown in Figure 3A. This finding suggests potential protection by specific IgA in tolerant patients, as IgA is generally able to prevent mucosal antigen crossing22. Regarding IgE, only a small number of V(D)J gene transcripts derived from IgE+ B-cells were successfully amplified (allergic: 1; tolerant: 2), possibly due to the extreme low abundance (0.002-1%) of IgE+ B-cells within the circulation23. Even though slightly higher proportions are known for IgG3+ (~1%) and IgG4+ B-cells (~0.75%)24, 25, no V(D)J gene transcripts derived from IgG3+ and IgG4+ B-cells were amplified. Overall, the individual isotype distribution varied for each patient, e.g. 2S albumin-binding B-cells of the allergic patient 4 were predominantly IgM+ B-cells, while those of patient 1 were predominantly IgG+ B-cells with a shift to class-switched B-cells in peanut allergic and tolerant patients. This indicates that the 2S albumin-binding B-cells from allergic and tolerant patients were more often matured and antigen-challenged B-cells compared with the non-atopic control group.