Beekeepers experience multiple bee stings each year. Many of these beekeepers (25-60%) become sensitized to bee venom through the production of specific antibodies that target the bee venom. Although these antibodies are important in the triggering of an allergic reaction only a small number of sensitised beekeepers go on to have an allergic reaction with symptoms away from the site of the sting. These reactions can be severe and are known as anaphylactic reactions.

The study investigates why some beekeepers develop severe allergic symptoms after bee stings while others do not.

This study will explore factors in the blood that protect sensitised individuals from having anaphylactic reactions - meaning that despite being sensitised they are tolerant and do not react to subsequent stings.

Beekeepers experience multiple stings each year many of whom (25-60%) become sensitised to bee (apis mellifera) venom through the production of IgE antibodies. However only a small proportion of the sensitised group go on to have anaphylaxis to bee venom. This study will investigate differences in T cell tolerance to bee venom in 3 groups by exploring factors that "protect" sensitised individuals from having anaphylactic reactions - meaning that despite being sensitised they are tolerant and do not react to subsequent stings.

T regulatory cell (Tregs) play a critical role in promoting immune tolerance to allergens and Treg generation has been shown to occur in bee venom allergic individuals following venom desensitisation therapy. Observations in sensitised tolerant (non-allergic) beekeepers (TB) suggest that exposure to venom through bee stings results in a similar increase in the frequencies of induced Tregs (iTregs). Helios negative induced Treg cells have also been suggested to contribute significantly to the establishment of immune tolerance in beekeepers. We will analyse the differences in Treg and Teffector (Teff) populations comparing sensitised beekeepers who are able to tolerate venom exposure (sensitised, tolerant beekeepers TB) with those beekeepers who do develop symptoms of systemic allergic reactions following bee sting (AB). A group of non-sensitised (NS) individuals will be recruited as a control population. We will perform RNA-seq to analyse the gene expression in Treg cells and Teff cells from all three groups. This will help to identify molecular targets that are involved in inducing the allergic reaction and also the genes that are involved in suppressing this reaction.

Considering the outcome of tolerance or anaphylaxis following bee stings, and the role of Treg and Teff cells in these processes, we will investigate the epigenetic regulations involved, which determine these cell's function. RNA-seq analysis will provide critical information about the genes involved in allergic reaction or in its suppression. DNA and histone modification analyses in the regulatory regions of these genes will help to better understand the underlying mechanism.

This project will investigate why some beekeepers who are sensitised to bee venom are able to tolerate bee stings, while in other sensitised individuals a bee sting can lead to life threatening anaphylaxis. This is not only of significance to beekeepers, but also has potential implications for the understanding of allergen tolerance in other allergic disease.

Inclusion Criteria:

- Group 1 inclusion criteria.

• Individuals naive to beekeeping (<2 previous bee stings, none in the last 24 months)
• no history of anaphylaxis to stings. Group 2 inclusion criteria.
• Beekeepers with > approximately 10 stings/year who have been beekeeping for >3 years.
• No history of anaphylaxis to bee venom. Group 3 inclusion criteria
• Beekeepers with diagnosis of anaphylaxis to bee venom in the last 12 months who are sensitised to bee venom (as evidenced by positive specific IgE and positive skin test to bee venom)

Exclusion Criteria:

• Unable to understand protocol/consent.
• Unable to commit to follow up visits.
• Participants with a known history of mastocytosis or under investigation for suspected mastocytosis.
• Participants undergoing desensitisation treatment to bee venom before the first visit.
• Participants being treatment with immunosuppressive medications. being treatment with immunosuppressive medications.

Group 1 inclusion criteria. Individuals naive to beekeeping (<2 previous bee stings, none in the last 24 months).

No history of anaphylaxis to bee stings.

Group 2 inclusion criteria. Beekeepers with > approximately 10 stings/year who have been beekeeping for >3 years.

No history of anaphylaxis to bee stings.

Group 3 inclusion criteria Beekeepers with diagnosis of anaphylaxis to bee venom in the last 12 months who are sensitised to bee venom (as evidenced by positive specific IgE and positive skin test to bee venom).