Background
Plasma extracellular vesicles (pEV) play a role modulating the immune response. In HIV/HCV coinfection, NK cells are crucial in eliminating activated hepatocellular stellate cells, responsible for progression to liver fibrosis (LF). We previously showed that in individuals with advanced LF (F4), NK cells presented an exhausted phenotype and impaired functionality even after elimination of HCV by direct-acting antivirals (DAA). Here, we aimed to characterize the effect on NK cell functionality of pEV from individuals with HIV/HCV and mild (METAVIR score F0/F1) or advanced LF (F4). We hypothesized that F4 pEV will have a negative impact on NK cells functionality.
Methods
pEV were purified by size exclusion chromatography from 2 mL of plasma from F0/F1 (n=5) and F4 (n=5) individuals at baseline (before DAA), EoT (end of treatment) and >= 1 year after successful treatment and from healthy controls (n=4). pEV were resuspended in 60 uL of PBS. PBMC were isolated from healthy donors and incubated for 1 hour with 30 uL of pEV suspension from each condition before conducting the NK cell functionality assay.
After pEV incubation, PBMC (1×106) were cocultured with K562 at a 10:1 ratio (1:1 NK:K562) for 2 hours. NK cell degranulation was evaluated by FACS analysis of CD107a, CD3, CD56 expression and a cell viability marker. We report NK relative degranulation as the ratio between NK CD107a+ in each condition and NK CD107a+ in absence of pEV. Data was analyzed using nonparametric statistics.
Results
We found that pEV from all participants, including healthy donors, consistently reduced NK CD107a expression, compared to NK cells in the absence of pEV. However, pEV from individuals with advanced liver fibrosis exhibited a higher suppressive effect on NK cell functionality than those from healthy donors (0.70 vs 0.88; p=0.03) and individuals with mild fibrosis at baseline (0.70 vs 0.85; p=0.03). The inhibitory effect of pEV on NK cell cytotoxicity was lower at EoT and at >= one year after EoT than baseline, for both groups.
Conclusions
We show evidence that pEV from individuals with advanced LF could play a role in the overall exhausted phenotype observed in NK cells from this group. This context could be one of the factors associated with little or no improvement of LF in individuals with F4 LF after eliminating HCV by DAA treatment. Further characterization of the composition of pEV could reveal therapeutic targets to reverse NK cell dysfunction and mitigate fibrosis in individuals with F4 LF.
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