Background: Although exposure to stings has been identified as the leading risk factor for anaphylaxis due to Hymenoptera venom allergy, professional beekeepers receive hundreds yearly without developing systemic reactions.
Objective: This study aims to analyse the mechanisms underlying bee venom tolerance in beekeepers.
Methods: A cross-sectional study was conducted. Participants were recruited and classified into three groups: allergic patients (AP) experiencing systemic reactions after bee stings, with a positive intradermal test and specific IgE (sIgE) to Apis mellifera venom (AmV); tolerant beekeepers (TBK) receiving ≥50 stings/year; and healthy non-exposed controls (HC). Serum levels of sIgE and specific IgG4 (sIgG4) to AmV, rApi m 1, rApi m 2, rApi m 3, Api m 4, rApi m 5 and rApi m10, as well as AmV-induced basophil degranulation, percentage of T-cell subsets, regulatory T-cells (Treg cells) and IL-10 production, were measured.
Results: APs had high levels of sIgE to AmV and all its allergic components (p<0.001) together with a high basophil activation rate (p<0.001) compared to TBKs. Conversely, compared to APs, TBKs showed higher levels of sIgG4 (p<0.001) and IL-10 (p<0.001) as well as an enhanced CTLA-4+ Treg population (p=0.001), expanded Helios- Treg (p<0.003), and reduced T-helper 1 (p=0.008), T-helper 2 (p=0.004) and T-helper 17 (p=0.007) subsets.
Conclusions: A different profile, strongly marked by Treg activity, was found in TBKs. This natural tolerance would be led by the expansion of inducible Helios-Treg cells at a peripheral level. Helios-Treg population could be a novel candidate biomarker useful for monitoring tolerance.

Key words: Helios protein, Regulatory T-cells, Immune tolerance, Bee venom allergy, Beekeeping