Fig 2: Effect of AA on the release of EVs. (A) Relative number of particles released by MDMs into the culture medium after 24 h of treatment with AA (blue dots) or solvent (grey dots) determined by HSFC. Values were calculated per mg total cell protein and normalized to 1 for solvent-treated cells. (B) Size distribution of particles released by MDMs shown in panel A. Histogram of particle size with a bin width of 10 nm for AA (blue bars) and solvent control (grey bars) samples. Data were normalized to the total number of acquired events separately for solvent and AA-treated samples. The plot shows the mean (± SEM) of the relative frequency of total events detected by HSFC in conditioned media from n = 5 donors. (C) Relative decrease of particle count in conditioned media after treatment with Triton X-100 (final concentration 0.05%). Values obtained before addition of detergent were set to 100%. (D) Immunostaining results on EV pellets obtained by differential centrifugation. The presence of tetraspanin markers (CD9, CD63 and CD81) was evaluated on particles obtained from n = 3-5 donors. A histogram of particle size of positive events is shown in Figure S8 for a representative sample. Bars represent mean (± SD) of total % of positive events. Symbols represent the values obtained with independent donors. Significance was tested by paired t test: * p < 0.05 for AA versus solvent.

Fig 3: Characterization of the EV fraction in the secretome of MenSCs. (a) EV release rate of MenSC under basal (white bars) and priming (gray bars) conditions. The number of particles released by MenSCs after 96 h was determined for the secretome samples (10,000× g supernatants) by nFC. Values were normalized to the total number of viable cells. (b) Median size (nm) of particles detected in EV-enriched fractions (EV-MenSCs) obtained after size exclusion chromatography (SEC) of the secretomes. (c) Histogram of particle size (SEC samples) with a bin width of 10 nm from primed (gray bars) and basal (white bars) MenSCs. Mean ± SD of the relative frequency of total events detected by nFC is shown. (d) Single-particle phenotyping of EV samples obtained by SEC. Three tetraspanin markers (CD9, CD63, and CD81) were evaluated by single-vesicle fluorescence analyses. Only those samples which showed positive staining were plotted. Different colored dots represent the values obtained with independent donors (n = 3). Significance was tested by a paired t-test. ** p < 0.01 in primed vs. basal conditions.

Fig 4: Location shapes the profile of intestinal CD103+CD69+CD8+T cells. (A) Volcano plot of the expressed genes, with a nominal P value <0.99, comparing intraepithelial to lamina propria (LP) CD8+CD69+CD103+ T cells; selected genes are highlighted. On the x-axis, the log2 fold change (log2FC) is shown, and on the y-axis, the -log10 P value (-log10P) is shown. Gray indicates not significantly differentially expressed genes, yellow indicates genes with a log2FC >0.25 and -log10P >10 × 10–2.5, green indicates genes with a log2FC > 0.25 and -log10P < 10 × 10–2.5. (B) Heatmap of the top 200 differentially expressed genes comparing CD103+ intraepithelial and lamina propria T cells with hierarchical clustering on the columns concerning compartment, status, and patient. Rows are z score normalized. (C) Pathway terms related to the 321 genes upregulated in intraepithelial (top) and 413 genes upregulated in LP (bottom) CD8+CD69+CD103+ T cells. (D) Messenger RNA (mRNA) expression (log2 counts; right) and percentage (left) of ileal CD8+CD69+CD103+ T cells expressing IL7R (CD127) in healthy control subjects (n = 3–5; diamonds) and CD patients from inflamed (circles) and noninflamed (triangles) ileum (paired, n = 4–5). Comparison was performed with Wald’s statistic and a paired 2-tailed t test, respectively. (E) Gene set enrichment analysis of Trm genes in humans (identified by Hombrink et al)25 in pairwise comparisons involving intraepithelial and LP CD8+CD69+CD103+ T cells derived from the ileum of healthy adult control subjects and CD patients pooled, represented by the normalized enrichment score and FDR statistical value (FDRq). (F) Representative gating strategy including Fluorescence Minus One (FMO) control (upper panel) and quantification (lower panel) of CD63 in CD8+CD69+CD103+ T cells comparing epithelium (IE) and LP. Bars represent median and interquartile range. Comparison was performed with a paired 1-tailed test. Symbols as per panel D. Healthy control subjects: n = 7; CD patients: n = 3–6. (G) As per panel F but for CXCR3. (H) As per panel F but for TIM-3 (left) and TIGIT (right). Comparison was performed with a paired 2-tailed t test. NS, not significant.

Fig 5: Platelet activation in mice systemically infected with C. albicans. (A) The number of circulating platelets in peripheral murine blood was determined with an automated hematology analyzer. Deposition of complement factor C3 (B), fibrinogen (C), and surface expression of CD63 (D) and CD62P (E) was quantified by flow cytometry. (F) Soluble CD62 was measured in plasma by ELISA. CTRL: Samples from non-infected control animals. Time on X axis: Time after infection. Four to seven samples were analyzed per time point, data is presented as individual data points with bars indicating the mean ± standard deviation. Asterisks indicate significant differences (p<0.05; 1-Way ANOVA and Holm-Sidak’s multiple comparison test to compare each infected group to the control).