Three of the five ‘classical’ HIES patients

had known STAT3 mutations (R382W twice and V463del) [5] (Table 1). Two of the patients with ‘classical’ HIES had no STAT3 mutation. To investigate the immunological functional properties Roxadustat with respect to Th17 responses in HIES patients with different mutations, PBMC from healthy volunteers, ‘classical’ HIES patients and three members from a HIES family with ‘variant’ HIES were assessed for the capacity to mount IL-17 responses. We have developed a new methodology of Th17 generation using human PBMC stimulated with whole microbial stimuli relevant for HIES: S. aureus and C. albicans[18]. HIES patients had a defective response to C. albicans, although IL-17 was measurable in all patients (Fig. 2a). Interestingly, IL-17 production was completely absent in PBMC stimulated with S. aureus in all ‘classical’ HIES patients (Fig. 2b). In contrast, PBMC isolated from the variant HIES patients, bearing the STAT3 mutations in the linker domain, were able to produce IL-17 in response to S. aureus, albeit at lower concentrations when compared to healthy volunteers (Fig. 2b and c). IFN-γ production was distorted in HIES patients when compared to healthy controls, while IL-10 was found to be elevated in HIES patients when stimulated with

both S. aureus and C. albicans. The in vitro stimulations described above suggest that HIES patients have a significant defect in the generation of Th17 cells. This was Hydroxychloroquine in vitro indeed the case for the patients with ‘classical’ HIES, either bearing STAT3 mutations or not (Fig. 3). Surprisingly, when the familial variant HIES patients were challenged with disease-related microorganisms, they showed a clear induction

of single IL-17-positive and IL-17/IFN-γ-positive CD4+ cells compared to normal controls (Fig. 3). IL-6 augmented IL-17 production induced by Immune system C. albicans and S. aureus in cells isolated from healthy controls (Fig. 4a). No effect was apparent in the HIES patients, independently of the type of STA3 mutation. In contrast to IL-6, IL-10 reduced the amount of IL-17, and this effect was observed both in healthy controls and HIES patients (Fig. 4b). Mutations in the SH2 and DNA-binding domain of STAT3 have been reported to be the cause of disease in a large proportion of HIES patients [4]. These mutations function as dominant-negative mutations [4] and result in a defective Th17 response in these patients [9,10], explaining many of the clinical features of HIES. In the present study we confirm, on one hand, the relationship between HIES and defective Th17 responses; on the other hand, we also refine this notion to include the relationships between the type of STAT3 mutation, immunological response to relevant microbial stimulation and clinical phenotype of the patients.