IL-10+ regulatory B cells are dysregulated in patients with seasonal and perennial allergy

B regulatory cells (Breg) have been shown to have the capacity to produce IL-10 and play a role in immune tolerance. We hypothesize that IL-35 supports the maintenance of immune tolerance by inducing IL-10+ Bregs. We further hypothesized that IL-10+ Bregs are dysregulated in individuals with seasonal and perennial allergy. Peripheral blood mononuclear cells (PBMCs) were collected from 30 subjects. PBMCs were stimulated with CpG and CD40L for 72 hours and induction of IL-10+ Breg cells were quantified by flow cytometry and the unbiased clustering tool FlowSOM. Level of secreted IL-10 was measured using ELISA. IL-10+ Breg cell subsets (IL-10+CD19+CD5+, IL-10+CD19+CD5hi and IL-10+ CD24hiCD38hi and IL-10+CD19+CD27+) were significantly induced in a time- and dose-dependent manner (non-atopic control (NAC), n=8; p<0.01). Recombinant IL-35 induced IL-10+ Breg cell subsets in the presence of CpG and CD40L in a concentration-dependent manner. FlowSOM analysis confirmed distinct metaclusters of IL-10+ Breg cells that were modulated by IL-35. A significant dysregulation in the proportion of all Breg cell subsets was observed in individuals with seasonal allergy (GPA, n=8) whilst individuals with perennial allergy displayed dysregulation in one specific Breg cell subset (IL-10+CD24hiCD38hi, n=10, p<0.05) compared to NAC. Unbiased machine learning tool FlowSOM revealed 2 specific metaclusters of dysregulated B cells found in both seasonal and perennial allergic patients compared to NAC. A trend of inverse correlation was observed between visual analogue score and IL-10+ Bregs. Our findings underscore the role of IL-35 in induction of IL-10+ Breg cells, which are dysregulated in seasonal and perennial allergics.