Single-particle analysis of small extracellular vesicles from the follicular fluid of women undergoing fertility treatments reveals distinct PD-L1+ populations

In some cell systems, small extracellular vesicles bearing PD-L1 (PD-L1+ sEVs) have been shown to be able to suppress T-cell immunity. We have herein investigated whether a distinct profile of PD-L1+ sEVs exists in human follicular fluid (FF). Single-particle interferometric reflectance imaging sensing combined with a single-particle antibody capture and immunofluorescence labelling were used to determine the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (n=10). In addition, the size distribution of sEVs was investigated via atomic force microscopy. Our data indicate that the bulk of tetraspanin-expressing EVs in human FF are less than 50 nm in size. Tetraspanins and PD-L1 exhibit distinct expression and colocalization profiles at sEV level across all cohort samples. A total of 42%, 46%, and 50% of all the particles captured by anti-CD63, anti-CD81, and anti-CD9 antibodies, respectively, were positive for CD81. PD-L1 was expressed at the highest level on CD9+ sEVs, with an average value of 5% within the cohort. The presence of distinct PD-L1+ sEV subpopulations suggests that they may play a role in regulating the immune response in the follicular microenvironment. Further research is needed to fully understand the functional significance of PD-L1+ sEVs in this context and their potential as biomarkers for predicting fertility outcomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/628903v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@77e67dorg.highwire.dtl.DTLVardef@1bd1ce2org.highwire.dtl.DTLVardef@b376d8org.highwire.dtl.DTLVardef@3f6fad_HPS_FORMAT_FIGEXP M_FIG C_FIG