Journal: bioRxiv

Article Title: Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

doi: 10.64898/2026.04.29.721601

Figure Lengend Snippet: Multiplex bead-based EV flow cytometry assay for surface markers. EVs enriched from UHPi and IVIg by SEC and dUC. Surface marker profiles were measured using Miltenyi MACSPlex EV kit IO with MESF-calibrated flow cytometry. (A) Heatmap and hierarchical clustering of MACSPlex markers in UHPi EVs, IVIg EVs, and bead/antibody controls (left). Median APC intensity (MESF, background-subtracted) of mixed tetraspanin antibodies (CD9/CD81/CD63) on EVs captured by 39 marker beads (middle/right). (B) Enlarged values for the tetraspanins (CD9, CD63, and CD81), HLA markers (HLA-ABC and HLA-DR, DP,DQ), platelet markers (CD42a, CD41b, and CD62p), and stemness markers (CD29, ROR1, CD24, CD326, CD133/1). dUC, differential ultracentrifugation; EV, extracellular vesicles; Human Leukocyte Antigens; HLA; IVIg, intravenous immunoglobulin; MESF, Molecular Equivalents of Soluble Fluorophore; SEC, size-exclusion chromatography; UHPi, individual unprocessed human plasma.

Article Snippet: Next, using the Cytex Amnis imaging flow cytometry high-resolution images of individual particles, we analyzed dUC- and SEC-isolated UHPp and IVIg EVs.

Techniques: Multiplex Assay, Flow Cytometry, Marker, Size-exclusion Chromatography, Clinical Proteomics

Journal: bioRxiv

Article Title: Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

doi: 10.64898/2026.04.29.721601

Figure Lengend Snippet: Flow cytometry phenotyping of CD63-positive EVs. UHPp, and IVIg EVs isolated using dUC or SEC were labeled with DiD and CD63-PE and analyzed by imaging flow cytometry. (A) Representative images of UHPp and IVIg EVs show morphology, BF, CD63, DiD, and scatter channels. (B) Scatter was used to gate out debris (left, gate R1). Fluorescent dot plots of unlabeled samples show background signal (middle) and labeled samples identified DiD + and CD63 + events (right, gate R2). (C) Summary plots of the frequency of EVs identified by scatter (gate R1, left) or fluorescence (gate R2, right) of UHPp, and IVIg EVs isolated using dUC or SEC. BF, bright field; DiD, 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; dUC, differential ultracentrifugation; EV, extracellular vesicles; IVIg, intravenous immunoglobulin; PE, phycoerythrin; SEC, size-exclusion chromatography; UHPp, pooled unprocessed human plasma. Note: two different lots of UHPp and IVIg were used for these experiments.

Article Snippet: Next, using the Cytex Amnis imaging flow cytometry high-resolution images of individual particles, we analyzed dUC- and SEC-isolated UHPp and IVIg EVs.

Techniques: Flow Cytometry, Isolation, Labeling, Imaging, Fluorescence, Size-exclusion Chromatography, Clinical Proteomics

Journal: bioRxiv

Article Title: Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

doi: 10.64898/2026.04.29.721601

Figure Lengend Snippet: IVIg EVs contain high levels of surface-associated IFNγ. (A) IVIg EV-rich fractions from SEC were subjected to proteinase K digestion for 1-30 min and levels of IFNγ and MIP-1β were analyzed by Luminex. (B) Flow cytometry analysis of different enrichment fractions. Particles were isolated from UHPi, SCIg, and IVIg by SEC. An EV gate was established based on size and lipophilic dye labeling with DiD. (C) Histograms depict representative fluorescence levels following labeling with an IFNγ antibody or isotype control. Summary plot contains MFI values of IFNγ (bars) or isotype (dashed line) staining of various fractions (E, EV-rich; P, protein-rich) isolated by dUC or SEC. Bars outline fractions containing EVs. DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; 4-chlorobenzenesulfonate salt; dUC, differential ultracentrifugation; EVs, extracellular vesicles; IFNγ, interferon gamma; IVIg, intravenous immunoglobulin; MFI, median fluorescence intensity; MIP-1β, macrophage inflammatory protein-1 beta; SEC, size-exclusion chromatography; UHPi, individual unprocessed human plasma.

Article Snippet: Next, using the Cytex Amnis imaging flow cytometry high-resolution images of individual particles, we analyzed dUC- and SEC-isolated UHPp and IVIg EVs.

Techniques: Luminex, Flow Cytometry, Isolation, Labeling, Fluorescence, Control, Staining, Size-exclusion Chromatography, Clinical Proteomics

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: CD34 + HSPC-derived neutrophils cultured in SL-II medium showed higher similarity to PMNs compared to CD34 + HSPC-derived neutrophils cultured in IMDM (A) Representative cytospins of PMNs, CD34 + HSPC-derived neutrophils cultured in SL-II (Bulk) and IMDM medium (Bulk) after Giemsa-May staining. Scalebars set at 10 μm, n = 14. (B) Gating strategy for comparing PMNs versus CD34 + HSPC-derived neutrophils cultured in SL-II or IMDM based on CD11b and CD16 expression as measured by flow cytometry. (C) Representative flow cytometric analysis of surface markers CD66b, CD29 and CD14 expressed on Bulk and CD16 high CD34 + HSPC-derived neutrophils cultured in either SL-II (pink) or IMDM medium (orange) and PMNs (blue). Gray histograms show unstained controls. n = 8 for CD66b, n = 4 for CD29 and CD14. (D) PCA plot showing transcriptomes and non-imputed proteomes for CD34 + HSPCs (yellow), SL-II Bulk obtained at days 14 (light pink) and 17 (black), IMDM Bulk obtained at day 14 (red) and 17 (purple), SL-II fractions MACS sorted for CD16 + obtained at day 14 (gray) and 17 (brown), and PMNs (blue). For SL-II Bulk day 17 transcriptomics n = 4, otherwise n = 6. (E) Pearson correlation between day 17 SL-II CD16 + neutrophils compared to CD34 + HSPCs and PMNs. Correlation values range between 0.6 and 1. (F) Heatmap of z-scores for transcriptome and proteome samples obtained from day 17 SL-II Bulk neutrophils, day 17 SL-II CD16 + neutrophils, PMNs, and CD34 + HSPCs. All transcripts or proteins that were considered differentially abundance between day 0 SL-II Bulk, day 10 SL-II Bulk, day 14 SL-II CD16 + neutrophils and day 17 SL-II CD16 + neutrophils when compared to PMNs were included. (G) Volcano plots of differentially expressed transcripts and proteins between day 17 SL-II CD16 + neutrophils compared to PMNs. (H) Functional enrichment showing the normalized enrichment score (NES) of molecular functions that were enriched within up- or downregulated transcripts and proteins in day 17 SL-II CD16 + neutrophils compared to PMNs. Molecular mechanisms were obtained from different databases, including Wiki-Pathways (∗), Gene Onthology (∗∗), or Reactome (∗∗∗). Neutrophil degranulation is highlighted in red. (I) Scatterplot comparing transcriptome and proteome effect size estimates for all transcript/protein pairs that were identified when comparing day 17 SL-II CD16 + neutrophils and PMNs. Gene/protein pairs highlighted in black were considered statistically significant after multiple testing correction FDR <0.05 and |log2 fold change| > 1 for proteome and 2 for transcriptome. n values represent the number of individual donor samples.

Article Snippet: We wish to thank the people from the Sanquin Central Facility, especially Erik Mul and Simon Tol, for helping us with setting up the imaging flow cytometry experiments.

Techniques: Derivative Assay, Cell Culture, Staining, Expressing, Flow Cytometry, Functional Assay

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: CD16 + cultured neutrophils were similar to PMNs in mobility, ROS production, phagocytosis, and microbial killing (A) Boxplots and violin plots showing the relative expression of genes and proteins involved in neutrophil effector functions . (B) CD11b/CD18-mediated adhesion assay of PMNs (blue) and SL-II CD16 + neutrophils (pink) to uncoated plastic plates ( n = 4). (C) Chemotactic potential of fluorescently labeled PMNs (blue) and SL-II CD16 + neutrophils (pink) based on movement through filters with a pore size of 3 micron ( n = 5). (D) NADPH oxidase assay to determine the production of extracellular peroxide after the addition of opsonized E. coli , zymosan, STZ, PMA, and PAF/fMLP of PMNs (blue) and SL-II CD16 + neutrophils (pink) ( n = 4 for SL-II CD16 + neutrophils and n = 8 for PMNs). (E) Phagocytosis of either unopsonized or opsonized zymosan by PMNs (blue) versus SL-II CD16 + neutrophils (pink) measured by flow cytometry ( n = 3 for SL-II CD16 + neutrophils and n = 4 for PMNs). (F) Representative images of phagocytosis of either unopsonized or opsonized zymosan labeled with FITC solution (green) by PMNs labeled with calcein red-orange (orange) versus SL-II CD16 + neutrophils labeled with calcein red-orange (orange) at the latest timepoint as visualized by imaging flow cytometric analysis. Scale bar was set at 10 μm ( n = 3). (G and H) Killing of opsonized E. coli and S. aureus, and unopsonized and opsonized C. albicans, respectively, shown for PMNs (blue) ( n = 3) versus SL-II CD16 + neutrophils (pink) ( n = 3). Killing was quantified as the inverse of colony-forming units (CFU) as a percentage relative to the CFU at the start of the assay. Negative values, signifying an increase in the number of colonies were considered to be 0. Data shown in (A–D, H) is represented as median and interquartile range, and (E) and (G) is represented as mean ± SD. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05 and ∗∗ p < 0.01. n values represent the number of individual donor samples.

Article Snippet: We wish to thank the people from the Sanquin Central Facility, especially Erik Mul and Simon Tol, for helping us with setting up the imaging flow cytometry experiments.

Techniques: Cell Culture, Expressing, Cell Adhesion Assay, Labeling, Pore Size, Flow Cytometry, Imaging, MANN-WHITNEY

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: SL-II CD16 + neutrophils retain tumor cell killing capacity but are deficient in immunosuppressive capacity (A) Cartoon of antibody dependent cellular cytotoxicity (ADCC) and myeloid derived suppressor cell (MDSC) activity exerted by mature activated neutrophils (Figure adapted from Aarts et al. ). (B) In vitro ADCC of LAN-1 cells unopsonized and opsonized (+Dinutuximab) ( n = 4) and SKBR3 cells unopsonized and opsonized (+Trastuzumab) by PMNs (blue) ( n = 6) and SL-II CD16 + neutrophils (pink) ( n = 3) in a 1:50 T:E ratio. (C) Representative CFSE plots for CD8 + T cell proliferation of an in vitro MDSC activity assay. T cells were stimulated with anti-CD3/CD28 antibodies to induce proliferation and co-cultured with either unstimulated or TNFα-stimulated PMNs or SL-II CD16 + neutrophils. After 4 days, T cell proliferation was assessed by CFSE dilution ( n = 12 for T cells alone and n = 16 for co-culturing with PMNs or SL-II CD16 + neutrophils). (D) Boxplots of CD8 + T cell proliferation and damaged T cell formation in the in vitro MDSC assay. Unstimulated or TNFα stimulated PMNs (blue) ( n = 12) or SL-II CD16 + neutrophils (pink) ( n = 16) were co-cultured with T cells. After 4 days, FSC/SSC gating was used to assess ‘damaged’ T cell formation next to T cell proliferation. (E) Representative images taken with imaging flow cytometry of trogocytosis carried out by PMNs and SL-II CD16 + neutrophils. The scale bar was set at 10 μm ( n = 3). PMNs and SL-II CD16 + neutrophils were stained with calcein red-orange (orange), and T cells were stained with DiD (red) before co-culturing for 4 h. Data in (B) and (D) is represented as median and interquartile range. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. n values represent the number of individual donor samples.

Article Snippet: We wish to thank the people from the Sanquin Central Facility, especially Erik Mul and Simon Tol, for helping us with setting up the imaging flow cytometry experiments.

Techniques: Derivative Assay, Activity Assay, In Vitro, Cell Culture, Imaging, Flow Cytometry, Staining, MANN-WHITNEY, Labeling

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: SL-II CD16 + neutrophils were able to release granules but showed granular content distinct from PMNs (A) Representative FSC/SSC plots measured by flow cytometry for PMNs and SL-II CD16 + neutrophils ( n = 14). (B) Release of serine-proteases ELANE and CTSG as measured by the maximal combined degradation of DQ-BSA after stimulation with CytoB and fMLP. Lysis of cells with Tx-100 was used as a control for maximal release. ( n = 4). (C) Geometric mean florescence intensity of CD63 (azurophilic granules) ( n = 10 for PMNs and n = 4 for SL-II CD16 + neutrophils), CD66b (specific granules) ( n = 9 for PMNs and n = 4 for SL-II CD16 + neutrophils) and LOX-1 (specific granules) ( n = 6 for PMNs and n = 4 for SL-II CD16 + neutrophils) on PMNs (blue) and SL-II CD16 + neutrophils (pink) in unstimulated conditions, after stimulation with PAF/fMLP or CytoB/fMLP measured by flow cytometry. Negative values were considered to be 0. (D) Representative histograms for degranulation (gMFI shown in C) by PMNs (blue) and SL-II CD16 + neutrophils (pink) under different conditions. Histograms of unstained controls are shown in gray. (E) Proportion of valid values for granule proteins for CD34 + HSPCs, CD16 + SL-II neutrophils, and PMNs. (F) Boxplots showing the log2 normalized read counts and log2 LFQ for signature granule proteins (i.e., MPO, ELANE, CTSG, LTF, and MMP9) for CD34 + HSPCs, CD16 + SL-II neutrophils, and PMNs. (G) Scatterplot of the effect size estimates for granule proteins when comparing CD16 + SL-II neutrophils to PMNs. Granule proteins were separated into azurophilic granules, specific granules, gelatinase granules, and secretory vesicles. Dots highlighted in red are considered statistically significant after multiple testing correction (FDR <0.05) and |log2 fold change| > 1. Data in (B), (C), and (F) is represented as median and interquartile range. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05, ∗∗ p < 0.01. n values represent the number of individual donor samples.

Article Snippet: We wish to thank the people from the Sanquin Central Facility, especially Erik Mul and Simon Tol, for helping us with setting up the imaging flow cytometry experiments.

Techniques: Flow Cytometry, Lysis, Control, MANN-WHITNEY, Labeling