Fig. 3.

EVs increase survival of cryptococci inside macrophages. a A schematic representation of the experimental assay in which extracellular vesicles (EVs) were added at different time points, either (i) to the cryptococci during opsonisation, 1 h prior to infection (‘Opsonisation’), (ii) directly to the macrophages J774 (MO J774) during 1 h of activation prior to infection (‘Activation’) or (iii) at the same time as the cryptococci are added to the macrophages (‘Infection’). b IPRs of R265 growing alone (R265), ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from R265 cells (EVs_R265) or heat-inactivated EVs_R265 (EVs_R265hk) added at different stages of infection, as described above: during yeast opsonisation by pooled human serum (‘PHS opsonisation’) or by GXM-specific antibodies—Mab 18B7 (‘Ab opsonisation’), J774 activation (‘Activation’) or during incubation with both macrophages and ICB180 yeast cells (‘Infection’). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 3 (with biological triplicates) to 11 independent experiments with 213–1767 total number of yeasts counted for each sample. Wilcoxon paired t test where * (P ≤ 0.05), significant difference; ** (P ≤ 0.01; and P = 0.0078 for infection with EVs_R265), highly significant difference and ns (P > 0.05), not significantly different. c IPR values can be further increased after adding higher amounts of EVs (+EVs_{R265-50 μg}). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 11 independent experiments with 1504–1948 total number of yeasts counted for each sample. Wilcoxon paired t test where * (P = 0.0186), significant difference. d EVs isolated from ICB180 (+EVs_ICB180) do not increase IPRs of ICB180 or R265 and proliferation of R265 is not enhanced by its own EVs (+EVs_R265) even at higher concentration (+EVs_{R265-50 μg}). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 9 to 24 independent experiments with 275–7888 total number of yeasts counted for each sample. Wilcoxon paired t test where ns (P > 0.05), not significantly different. e IPR of a non-virulent ICB180 strain is not altered by the presence of EVs isolated from C. neoformans virulent strain KN99 even at higher concentration of those vesicles (+EVs_{KN99-50 μg}) added during the infection step. Data are presented as scattered dot plots with lines representing their medians. Individual Wilcoxon matched-pairs signed rank test presented as P values above each dot plot, where ns (P > 0.05), not significantly different. Data are representative of results from at least 16 independent experiments with 1742–5209 total number of yeasts counted for each sample