Table 1.
Characterisation of study subjects.
Figure 1.
M. sympodialis releases extracellular vesicles.
(A) M. sympodialis culture supernatants were ultracentrifuged and sucrose gradient fractions coated onto latex beads and analysed for the presence of M. sympodialis epitopes using flow cytometry. Data is from one representative out of 6 independent experiments and displayed as mean MFI ratio of a rabbit antibody against M. sympodialis and unspecific rabbit IgG. Fractions ranging in density from 1.11–1.20 g/ml were pooled and analysed by (B) TEM and (C) IEM with the same antibody as in A. Scale bars correspond to 100 nm. Pictures are representative in (B) of 4 independent experiments and in (C) of two independent experiments. (D) Western blot analysis of sucrose gradient fractions analysed using a rabbit antibody against M. sympodialis and (E) using serum from an AE patient sensitized to M. sympodialis to detect IgE-binding epitopes. Data shown in (D) and (E) are from one out of 2 experiments using separate sucrose gradients.
Figure 2.
MalaEx elicit IL-4 and TNF-α responses.
(A) IL-4 production in CD14, CD34 depleted PBMC as measured by ELISPOT from HC and AE patients when cultured alone (medium) or with MalaEx or M. sympodialis (Mala) for 48 hr. (B) TNF-α levels in the IL-4 ELISPOT supernatants were measured by ELISA. Data represent mean values of triplicates for each individual (Table 1); lines indicate median.
Figure 3.
DCexo Mala contain M. sympodialis antigens.
(A) Sucrose gradient fractions of exosomes from MDDC cultured alone (DCexo) or with M. sympodialis (DCexo Mala) were analysed for the expression of HLA-DR and CD63 using flow cytometry. Data are representative for one out of 3 experiments each using a pool of exosomes from 2 healthy blood donors and displayed as MFI ratio between specific antibody and isotype control. (B) Sucrose gradient fractions of DCexo and DCexo Mala analysed using flow cytometry. Data are from 5 independent experiments and displayed as mean MFI ratio ± SEM between a rabbit antibody against M. sympodialis and unspecific rabbit IgG. (C) Double IEM was performed on pools of DCexo and DCexo Mala from 5 AE patients and 3 HC as well as 2 independent preparations of MalaEx using a mAb against HLA-DR (detected with a 5 nm gold-particle conjugated goat anti-mouse Ab, white arrows) and a rabbit antibody against M. sympodialis (detected with a 10 nm gold-particle conjugated goat anti-rabbit Ab, black arrows) and the corresponding isotype controls. Scale bars correspond to 100 nm.
Figure 4.
DCexo Mala elicit IL-4 and TNF-α responses.
(A) IL-4 production in autologous CD14, CD34 depleted PBMC from AE patients and HC as measured by ELISPOT when cultured alone (medium), with DCexo, DCexo Mala, M. sympodialis (Mala) or MDDC co-cultured with M. sympodialis (DCMala) for 48 hr. (B) TNF-α levels in the IL-4 ELISPOT supernatants were measured by ELISA. Data represent mean values of triplicates for each individual (Table 1); lines indicate median. (C) TNF-α levels in supernatants of CD14, CD34 depleted PBMC of two additional healthy blood donors (HC A and HC B) when cultured alone (un), with DCexo or DCexo Mala (Exo) or with DCexo and DCexo Mala that were depleted of MHC class II positive vesicles (depl. Exo). Bars indicate mean value of duplicates.
Figure 5.
Plasma exosomes elicit TNF-α but not IL-4 responses.
(A) Sucrose gradient fractions of plasma exosomes pooled from 2 healthy blood donors were loaded onto anti-MHC class II Dynabeads and analysed for the presence of HLA-DR and CD81 using flow cytometry. MFI ratio was calculated by dividing the sample MFI by the isotype control MFI. Data are from one out of two experiments. (B) plasma exosomes isolated from HC and AE patients were loaded onto anti-MHC class II Dynabeads and phenotyped using flow cytometry. Lines indicate the median. (C) PBMC were either cultured in only medium in the absence (−) or presence (+) of M. sympodialis or with autologous plasma exosomes in the absence (−) or presence (+) of M. sympodialis. IL-4 production in PBMC were measured by ELISPOT. (D) Culture supernatants from (C) were analysed for TNF-α levels using ELISA. Data represent mean values of triplicates for each individual (Table 1); lines indicate median.