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Journal Articles

CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy

Open Access
Paola Lanuti, Francesco Guardalupi, Giulia Corradi, Rosalba Florio, Davide Brocco, Serena Veschi, Elsa Pennese, Domenico De Bellis, Francesca D’Ascanio, Anna Piro, Laura De Lellis, Pasquale Simeone, Maria Concetta Cufaro, Serena Pilato, Isabella D’Amario, Ida Villanova, Barbara Di Francesco, Lucia Di Re, Fabio Verginelli, Damiana Pieragostino, Prassede Salutari, Fabrizia Colasante, Annalisa Natale, Maria Vittoria Mattoli, Simone Vespa, Antonella Fontana, Raffaella Giancola, Bianca Fabi, Stefano Baldoni, Stella Santarone, Francesco Restuccia, Nicola Tinari, Piero Del Boccio, Alessandro Cama, Mauro Di Ianni
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2907–2919.
https://doi.org/10.1182/bloodadvances.2024014860
Published: 2025
Includes: Supplemental data
Journal Articles

Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia

Open Access
Alexander Dupuy, Xiaoming Liu, Yvonne Kong, Miao Qi, Jose Perdomo, Jemma Fenwick, Jessica Tieng, Bede Johnston, Qiyu Sara Shi, Mark Larance, Yingqi Zhang, Lining Arnold Ju, Paul Coleman, Jennifer Gamble, Elizabeth E. Gardiner, Mortimer Poncz, Huyen Tran, Vivien Chen, Freda H. Passam
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2891–2906.
https://doi.org/10.1182/bloodadvances.2024014165
Published: 2025
Includes: Multimedia, Supplemental data
Journal Articles

Hematopoietic progenitor kinase-1 inhibition improves the in vitro efficacy of bispecific antibodies in CLL

Open Access
Yamit Shorer Arbel, Liron Yitzhak, Ben-Zion Katz, Yair Herishanu
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2922–2926.
https://doi.org/10.1182/bloodadvances.2024015242
Published: 2025
Includes: Multimedia, Supplemental data
Journal Articles

Prognostic role of interim PET in follicular lymphoma: a post hoc study of FOLL12 trial by Fondazione Italiana Linfomi

Open Access
Clinical Trials & Observations
Rexhep Durmo, Stephane Chauvie, Federico Fallanca, Fabrizio Bergesio, Antonio Pinto, Ilaria Del Giudice, Marta Coscia, Paolo Corradini, Emanuele Angelucci, Patrizia Tosi, Roberto Freilone, Filippo Ballerini, Alessia Bari, Domenico Pastore, Pier Luigi Zinzani, Silvia Bolis, Leonardo Flenghi, Arcangelo Liso, Jacopo Olivieri, Luigi Marcheselli, Michele Merli, Annibale Versari, Luca Guerra, Stefano Luminari
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2927–2934.
https://doi.org/10.1182/bloodadvances.2024014790
Published: 2025
Includes: Supplemental data
Journal Articles

Spatial transcriptomics of progression gene signature and tumor microenvironment leading to progression in mycosis fungoides

Open Access
Myoung Eun Choi, Gyeonghoon Kim, Hwa-Jeong Shin, Chong Hyun Won, Sung Eun Chang, Mi Woo Lee, Woo Jin Lee
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2871–2885.
https://doi.org/10.1182/bloodadvances.2024014495
Published: 2025
Includes: Supplemental data
Journal Articles

AhR activation mitigates graft-versus-host disease of the central nervous system by reducing microglial NF-κB signaling

Open Access
Alexander Zähringer, Inês Morgado, Daniel Erny, Florian Ingelfinger, Jana Gawron, Sangya Chatterjee, Valentin Wenger, Dominik Schmidt, Lennard Schwöbel, Rachael C. Adams, Marlene Langenbach, Alina Hartmann, Natascha Osswald, Julian Wolf, Günther Schlunck, Priscilla S. Briquez, Kathleen Grueter, Dietrich A. Ruess, Ian Frew, Ann-Cathrin Burk, Verena Holzmüller, Bodo Grimbacher, David Michonneau, Geoffroy Andrieux, Gérard Socié, Julia Kolter, Melanie Boerries, Marie Follo, Franziska Blaeschke, Lisa Sevenich, Marco Prinz, Robert Zeiser, Janaki Manoja Vinnakota
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2935–2952.
https://doi.org/10.1182/bloodadvances.2024015000
Published: 2025
Includes: Supplemental data
Journal Articles

The role of miR-150-5p/E2F3/survivin axis in the pathogenesis of plasmablastic lymphoma and its therapeutic potential

Open Access
Miriam Verdú-Bou, Maria Joao Baptista, Marcelo Lima Ribeiro, Aleix Méndez-López, Núria Profitós-Pelejà, Fabian Frontzek, Gaël Roué, José Luís Mate, Mireia Pellicer, Pau Abrisqueta, Josep Castellví, Mariana Bastos-Oreiro, Javier Menárguez, Miguel Alcoceba, Eva González-Barca, Fina Climent, Antonio Salar, Juan-Manuel Sancho, Annette M. Staiger, German Ott, Ioannis Anagnostopoulos, Manel Esteller, Georg Lenz, Gustavo Tapia, José-Tomás Navarro
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2953–2967.
https://doi.org/10.1182/bloodadvances.2025016180
Published: 2025
Includes: Supplemental data
Journal Articles

Extracellular vesicles for CAR T-cell therapy immunomonitoring

Open Access
Marion Klea Pinheiro, Benoît Vingert
Journal: Blood Advances
Blood Adv (2025) 9 (12): 2920–2921.
https://doi.org/10.1182/bloodadvances.2025015988
Published: 2025
Images
Monitoring of CD19 CAR T cells and CD19.CAR+EVs in patients treated with CD19 CAR T cells. A total of 22 patients were monitored for 2 years from CD19 CAR T-cell infusion. The concentration of circulating CD19 CAR T cells was monitored both by flow cytometry (purple line) and ddPCR (blue line). CD19 CAR T-cell monitoring was paralleled with the concentration of CD19.CAR+EVs (red line). Flow cytometry analysis of CD19.CAR+EVs in responding vs nonresponding patients is also shown. Data were log transformed. A 2-way analysis of variance (ANOVA), followed by Tukey multiple comparisons test, was performed. An alpha value of .05 was established for the significance threshold. Each time point represents the mean (+SEM) of at least 3 independent measurements from distinct patients. Data are represented as mean + SEM; Mann-Whitney nonparametric test, and Robust Regression followed by OUTlier Identification test for outliers were applied.

Monitoring of CD19 CAR T cells and CD19.CAR + EVs in patients treated with ...

Open Access
in CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy > Blood Advances
Published: 2025
Figure 1. Monitoring of CD19 CAR T cells and CD19.CAR + EVs in patients treated with CD19 CAR T cells. A total of 22 patients were monitored for 2 years from CD19 CAR T-cell infusion. The concentration of circulating CD19 CAR T cells was monitored both by flow cytometry (purple line) and ddPCR (... More about this image found in Monitoring of CD19 CAR T cells and CD19.CAR + EVs in patients treated with ...
Images
Characterization of CD19.CAR+EVs. (A) NTA (i); AFM analysis of circulating CD19.CAR+EVs (ii); western blot analysis of CD63, flotillin-1, and cytochrome C in circulating CD19.CAR+EV samples (T lymphocytes were used as controls for cytochrome C expression) (iii); and flow cytometry evaluation of CD63 and flotillin-1 expression in circulating CD19.CAR+EVs (iv). CD63 and flotillin-1 expression (red histograms) were analyzed using the related fluorescence minus one (FMO) as controls (blue histograms). Mean fluorescence intensity (MFI) ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments. (B) Transmission electron microscopy analysis of circulating CD19.CAR+EVs (scale bar, 100 nm). (C) Flow cytometry evaluation of CAR expression on circulating CD19.CAR+EVs. CAR expression (red histogram) was analyzed using the related FMOs as controls (blue histogram). MFI ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments. (D) Venn diagram shows the common and uncommon proteins identified in circulating and preinfusion CD19.CAR+EVs. Same amount of EVs (pool of 3 individuals per condition containing 3 × 106 EVs) were compared when proteomic analyses were performed to parallel circulating vs preinfusion EV cargoes. The number 4 refers to the number of identified proteins (4, corresponding to the 1.41% of the total number of identified proteins) uniquely carried by circulating EVs, whereas 275 proteins (99.97 % of identified proteins) were those shared by circulating and preinfusion CAR+EVs, and 4 other proteins (1.41% of identified proteins) were carried only by preinfusion CAR+EVs. (E) NTA (i) and AFM analysis (ii) of preinfusion CD19.CAR+EVs; western blot analysis of CD63, flotillin-1, and cytochrome C in preinfusion CD19.CAR+EV samples (T lymphocytes were used as controls for cytochrome C expression) (iii); and flow cytometry evaluation of CD63 and flotillin-1 expression in preinfusion CD19.CAR+EVs (iv). CD63 and flotillin-1 expression (red histograms) were analyzed using the related FMOs as controls (blue histograms). MFI ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments.

Characterization of CD19.CAR + EVs. (A) NTA (i); AFM analysis of circulati...

Open Access
in CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy > Blood Advances
Published: 2025
Figure 2. Characterization of CD19.CAR + EVs. (A) NTA (i); AFM analysis of circulating CD19.CAR + EVs (ii); western blot analysis of CD63, flotillin-1, and cytochrome C in circulating CD19.CAR + EV samples (T lymphocytes were used as controls for cytochrome C expression) (iii); and flow cytometr... More about this image found in Characterization of CD19.CAR + EVs. (A) NTA (i); AFM analysis of circulati...
Images
In vitro cytolytic activity of CD19.CAR+EVs on Raji and SUP-B15 cell lines. (A) Cell viability of 2 cell lines, Raji (i) and SUP-B15 (ii), assessed by MTT assays after incubation for 48 hours with CD19.CAR+EVs at the indicated concentrations. Data shown are means ± SD of 3 to 4 replicates. ∗Statistically significant differences, compared with control (0 μg); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (B) Flow cytometry killing assays were performed using the concentration of 0.015 μg of circulating EVs per target cell to treat both Raji and SUP-B15 cell lines and analyzing their cytolytic activity by measuring the 7-AAD staining of target cells after 24 hours of treatment (Student t test, P = .0187). Values are averages of 3 independent experiments. (C) Flow cytometry killing assays were performed using the concentration of 0.015 μg of preinfusion EVs per target cell to treat both Raji and SUP-B15 cell lines and analyzing their cytolytic activity by measuring the 7-AAD staining of target cells after 24 hours of treatment (Student t test, not significant). (D) The immunogenicity of CD19.CAR+EVs was studied by treating heterologous T cells for 24 hours with the same dose of CD19.CAR+EVs used for testing their cytolytic abilities (0.015-μg protein EV per target cell). The red histogram represents the CFSE profile of untreated heterologous T cells, whereas the overlaid blue histogram represents the CFSE profile of heterologous T cells treated with CD19.CAR+EVs. Data are representative of 2 independent experiments. CFSE, carboxyfluorescein succinimidyl ester.

In vitro cytolytic activity of CD19.CAR + EVs on Raji and SUP-B15 cell line...

Open Access
in CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy > Blood Advances
Published: 2025
Figure 3. In vitro cytolytic activity of CD19.CAR + EVs on Raji and SUP-B15 cell lines. (A) Cell viability of 2 cell lines, Raji (i) and SUP-B15 (ii), assessed by MTT assays after incubation for 48 hours with CD19.CAR + EVs at the indicated concentrations. Data shown are means ± SD of 3 to 4 rep... More about this image found in In vitro cytolytic activity of CD19.CAR + EVs on Raji and SUP-B15 cell line...
Images
CD19.CAR+EV protein cargo. (A) Estimated MEFs per μm2. The analysis of MEF was performed both on circulating CD19.CAR+EVs and their circulating parental cells from the same patients (n = 3). The red dots refer to the estimated MEFs per μm2 of CD19.CAR+EV surface, whereas the blue dots represents the estimated MEFs per μm2 of CAR T-cell surface. (B) Dot plots showing expression of granzyme B and perforin in circulating CD19.CAR+EVs; the fluorescence minus one (FMO) control for granzyme B is shown (i); the percentage of granzyme B–positive EVs is represented (mean percent, 61.9% [SD, 4.7%]; mean MFI ratio, 3.5 [SD, 0.8]) (ii); the control FMO of perforin is shown (iii); and the percentage of perforin-positive EVs is represented (mean percent, 67.6% [SD, 4.2%]; mean MFI ratio, 6.7 [SD, 1.3]) (iv). The protein functional analyses of circulating (C) and preinfusion CD19.CAR+EVs (D), calculated by IPA with all identified proteins, are shown. IPA, ingenuity pathway analysis.

CD19.CAR + EV protein cargo. (A) Estimated MEFs per μm 2 . The analysis of...

Open Access
in CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy > Blood Advances
Published: 2025
Figure 4. CD19.CAR + EV protein cargo. (A) Estimated MEFs per μm 2 . The analysis of MEF was performed both on circulating CD19.CAR + EVs and their circulating parental cells from the same patients (n = 3). The red dots refer to the estimated MEFs per μm 2 of CD19.CAR + EV surface, whereas the ... More about this image found in CD19.CAR + EV protein cargo. (A) Estimated MEFs per μm 2 . The analysis of...
Images
CD19 CAR T-cell–expressing PD-1 and LAG-3 are functional and produce EVs. (A) Flow cytometry analysis of markers, known to be involved in T-cell exhaustion, expressed on CD19 CAR T cells, residual from the infusion bags. The 3 graphs indicate the percentage of PD-1+ cells, LAG-3+ cells, and TIM-3+ cells gated on total CD3+ CAR T cells (gray circles), CD4+ CAR T cells (blue circles), and CD8+ CAR T cells (blue circles). Data are presented as mean ± SEM (1-way ANOVA; CD4+PD-1+ CAR T cells vs CD8+PD-1+ CAR T cells, P = .0100; CD4+LAG-3+ CAR T cells vs CD8+LAG-3+ CAR T cells, P = .0386). (B) Cytotoxic function of total CD3+ CAR T cells, CD4+PD-1+ CAR T cells, CD4+PD-1– CAR T cells, CD8+LAG-3+ CAR T cells, and CD8+LAG-3– CAR T cells, isolated by FACS from the infusion bag, against a CD19-expressing cell line, Raji. After 24 hours of culture, the percentage of target living cells with CAR T cells (target-to-CAR T-cell ratio, 1:10) was assessed by flow cytometry. The bars represent the mean and SEM of killing of CAR T cells derived from at least 4 different donors. (Student t test; CD3+ CAR T cells vs CD8+LAG-3+ CAR T cells, P = .0115; CD4+PD-1+ CAR T cells vs CD8+LAG-3+ CAR T cells, P = .0160; CD4+PD-1– CAR T cells vs CD8+LAG-3+ CAR T cells, P = .0137). (C) Comparison of the percentage of CD4+PD-1+ CAR T cells and CD8+LAG-3+ CAR T cells, assessed by flow cytometry. The cells analyzed were obtained from the residual bags or from the PB of patients, 14 days after CD19 CAR T-cell infusion (2-way ANOVA, not significant). (D) The protein functional analysis calculated by ingenuity pathway analysis with all identified proteins is shown for CD8+LAG3+ EVs paralleled with the CD8+LAG-3– EV compartment.

CD19 CAR T-cell–expressing PD-1 and LAG-3 are functional and produce EVs. ...

Open Access
in CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy > Blood Advances
Published: 2025
Figure 5. CD19 CAR T-cell–expressing PD-1 and LAG-3 are functional and produce EVs. (A) Flow cytometry analysis of markers, known to be involved in T-cell exhaustion, expressed on CD19 CAR T cells, residual from the infusion bags. The 3 graphs indicate the percentage of PD-1 + cells, LAG-3 + c... More about this image found in CD19 CAR T-cell–expressing PD-1 and LAG-3 are functional and produce EVs. ...
Images
Characterization of CD3+–immune-selected CD19.CAR+EVs. NTA (A) and AFM analysis (B). (C) Flow cytometry evaluation of CD63 and flotillin-1 expression. CD63 and flotillin-1 expression (red histograms) were analyzed using the related FMOs as controls (blue histograms). MFI ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments. (D) Flow cytometry killing assays were performed using the concentration of 0.015 μg of protein from CD3-immunoselected EVs per target cell to treat both Raji and SUP-B15 cell lines and by analyzing their cytolytic activity by measuring the 7-AAD staining of target cells after 24 hours of treatment.

Characterization of CD3 + –immune-selected CD19.CAR + EVs. NTA (A) and AFM...

Open Access
in CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy > Blood Advances
Published: 2025
Figure 6. Characterization of CD3 + –immune-selected CD19.CAR + EVs. NTA (A) and AFM analysis (B). (C) Flow cytometry evaluation of CD63 and flotillin-1 expression. CD63 and flotillin-1 expression (red histograms) were analyzed using the related FMOs as controls (blue histograms). MFI ratio valu... More about this image found in Characterization of CD3 + –immune-selected CD19.CAR + EVs. NTA (A) and AFM...
Images
Endo-chip assay. (A) Endothelial cells (HUVECs) are seeded into the microchannel of the chip and allowed to adhere for 4 to 12 hours. Inset on the left shows differential interference contrast image of cells coating the channel. Test sample (eg, patient serum) is injected into the channel and incubated with endothelial cells for 30 minutes and then washed off. Venous blood from a healthy donor is collected into a citrate tube. Fluorescent antibodies are added to the blood (to label platelets, neutrophils, and fibrin). Immediately before perfusion, the whole blood is recalcified. The blood is perfused over the HUVEC layer and fluorescent images are captured by confocal microscopy. Inset on the right shows a representative image of the channel after perfusion of blood showing platelet, fibrin, and neutrophil adhesion to the endothelial layer (thromboinflammation). Image created with BioRender.com. (B) Accumulation of platelets, neutrophils, and fibrin after 15-minute perfusion of blood on HUVECs treated with media, ChAdOx1 nCOV-19, or TNF-α (5 ng/mL, positive control). Representative images of platelet (green), neutrophil (red), and fibrin (magenta) merged images after perfusion of blood. (C) Surface area coverage per field of platelets, number of neutrophils per field, and surface area coverage per field of fibrin at the end of a 15-minute perfusion of blood on HUVECs treated with media, ChAdOx1 nCOV-19, or TNF-α (5 ng/mL). Mean ± standard deviation (SD) of n = 3 independent experiments. One-way analysis of variance (ANOVA) with Tukey post hoc test was used for comparisons. (D) Kinetics of the platelet, neutrophil, and fibrin accumulation on the endothelial surface over 15 minutes of perfusion.

Endo-chip assay. (A) Endothelial cells (HUVECs) are seeded into the microc...

Open Access
in Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia > Blood Advances
Published: 2025
Figure 1. Endo-chip assay. (A) Endothelial cells (HUVECs) are seeded into the microchannel of the chip and allowed to adhere for 4 to 12 hours. Inset on the left shows differential interference contrast image of cells coating the channel. Test sample (eg, patient serum) is injected into the chan... More about this image found in Endo-chip assay. (A) Endothelial cells (HUVECs) are seeded into the microc...
Images
Endothelial thromboinflammation develops in the Endo-chip in response to both VITT ELISA positive and negative sera. (A) Optical density (OD) of VITT samples that tested positive by routine PF4/polyanion ELISA (VITT ELISA positive), and negative by routine PF4/polyanion ELISA (VITT ELISA negative). The OD was also measured for samples from patients with VTE without VITT and in vax controls. The dotted black lines represent the lowest and highest cutoff for positivity determined by the manufacturer. (B) Platelet (green), neutrophil (red), and fibrin (purple) accumulation following a 15-minute blood perfusion in the Endo-chip after treatment with VITT ELISA positive, VITT ELISA negative, VTE without VITT, or vax control sera. Representative images. (C) Platelet fluorescence area per field, neutrophil count per field, and fibrin fluorescence area per field following a 15-minute perfusion of blood in the Endo-chip after treatment with VITT ELISA positive, VITT ELISA negative, VTE without VITT, or vax control sera. VITT ELISA positive n = 15, VITT ELISA negative n = 17, VTE no VITT n = 8, and vax control n = 15. The individual patient numbers are shown in panel A. One-way ANOVA with Dunn post hoc test was used for comparisons.

Endothelial thromboinflammation develops in the Endo-chip in response to bo...

Open Access
in Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia > Blood Advances
Published: 2025
Figure 2. Endothelial thromboinflammation develops in the Endo-chip in response to both VITT ELISA positive and negative sera. (A) Optical density (OD) of VITT samples that tested positive by routine PF4/polyanion ELISA (VITT ELISA positive), and negative by routine PF4/polyanion ELISA (VITT ELI... More about this image found in Endothelial thromboinflammation develops in the Endo-chip in response to bo...
Images
PF4 enhances endothelial thromboinflammation induced by VITT serum in the Endo-chip. (A) Serum concentration of PF4 (ng/mL) in VITT ELISA positive, VITT ELISA negative, VTE without VITT, and vax control samples. One-way ANOVA with Tukey post hoc test was used for comparison. (B) Platelet (green), neutrophil (red), and fibrin (purple) accumulation following a 15-minute blood perfusion in the Endo-chip after treatment with VITT ELISA positive, VITT ELISA negative, or vax control serum without (upper panel) or with (lower panel) the addition of PF4. Representative images. (C) Platelet fluorescence area per field, neutrophil count per field, and fibrin fluorescence area per field following a 15-minute blood perfusion in the Endo-chip after treatment with VITT ELISA positive serum without or with addition of PF4, VITT ELISA negative serum without or with addition of PF4, and vax control serum without or with addition of PF4. A paired t test was used for comparison. (D) Kinetics of the platelet fluorescence area, neutrophil count, and fibrin fluorescence area over 15 minutes of blood perfusion in the Endo-chip after treatment with VITT ELISA positive serum without or with addition of PF4, VITT ELISA negative serum without or with addition of PF4, or vax control serum without or with addition of PF4. VITT ELISA positive n = 7 (patients 1, 2, 4, 5, 6, 8, 11), VITT ELISA negative n = 11 (patients 1, 2, 4, 5, 6, 8, 9, 10, 11, 12, 14), vax controls n = 11 (patients 1, 2, 5, 6, 7, 8, 10, 12, 13, 14, 15). PF4 was added at 25 μg/mL in all experiments.

PF4 enhances endothelial thromboinflammation induced by VITT serum in the E...

Open Access
in Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia > Blood Advances
Published: 2025
Figure 3. PF4 enhances endothelial thromboinflammation induced by VITT serum in the Endo-chip. (A) Serum concentration of PF4 (ng/mL) in VITT ELISA positive, VITT ELISA negative, VTE without VITT, and vax control samples. One-way ANOVA with Tukey post hoc test was used for comparison. (B) Platel... More about this image found in PF4 enhances endothelial thromboinflammation induced by VITT serum in the E...
Images
VITT serum induces endothelial cell activation, which is enhanced with the addition of PF4. (A) Immunostaining of the HUVEC layer for spike protein (red), nuclei, TF, and P-selectin (lower panel) after incubation with media alone or with media containing ChAdOx1 nCOV-19 adenoviral vaccine. (B) Immunostaining for human IgG (green) in HUVECs after treatment with VITT sera vs vax control sera. Representative images. The fluorescence intensity of IgG per field is shown for VITT and vax controls. Mean ± SD from 3 to 5 fields of view; n = 4 independent experiments, unpaired t test. (C) Representative images of HUVECs stained for TF (green), P-selectin (red), and VCAM-1 (magenta) after exposure to TNF-α (5 ng/mL), VITT serum, VITT serum + PF4 (25 μg/mL), vax control serum, vax control serum + PF4 (25 μg/mL), or media. Nuclear staining using Hoechst is shown in blue. (D) Fluorescence intensity of TF, P-selectin, and VCAM-1 of HUVECS treated with VITT, without or with 25 μg/mL PF4 added, or treated with vax control, without or with 25 μg/mL PF4 added, expressed as fold change in comparison with the fluorescent intensity of media alone. Mean ± SD, from 3 to 5 fields of view; VITT ELISA positive n = 2 (patients 2, 4), VITT ELISA negative n = 2 (patients 3, 9), vax control n = 4 (patients 2, 6, 10, 12). One-way ANOVA with Dunn post hoc test was used for comparison. (E) Fold change in the mRNA expression of F3 (TF) in HUVECs treated with plasma, with or without PF4 (25 μg/mL), in comparison with that of glyceraldehyde-3-phosphate dehydrogenase. Mean ± SD, n = 10 for media, VITT ELISA positive n = 21 (Table 1), VITT ELISA negative n = 17 (Table 1), vax control n = 8 plasma samples. One-way ANOVA with Dunn post hoc test was used for comparisons.

VITT serum induces endothelial cell activation, which is enhanced with the ...

Open Access
in Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia > Blood Advances
Published: 2025
Figure 4. VITT serum induces endothelial cell activation, which is enhanced with the addition of PF4. (A) Immunostaining of the HUVEC layer for spike protein (red), nuclei, TF, and P-selectin (lower panel) after incubation with media alone or with media containing ChAdOx1 nCOV-19 adenoviral vacc... More about this image found in VITT serum induces endothelial cell activation, which is enhanced with the ...
Images
The endothelial thromboinflammatory response in VITT is antibody dependent. (A) Platelet fluorescence area per field, neutrophil count per field, and fibrin fluorescence area per field following a 15-minute blood perfusion in the Endo-chip after treatment with VITT ELISA positive serum or IgG depleted serum (upper panel) or with VITT ELISA negative serum or IgG depleted serum (lower panel) in the presence or absence of 25 μg/mL PF4. VITT ELISA positive n = 5 (patients 2, 3, 4, 6, 12), VITT ELISA negative n = 5 (patients 1, 3, 4, 5, 6). One-way ANOVA with Dunn post hoc test was used for comparisons. (B) Platelet fluorescence area per field, neutrophil count per field, and fibrin fluorescence area per field following a 15-minute blood perfusion in the Endo-chip after treatment with IgG isolated from the same VITT ELISA positive serum or VITT ELISA negative serum as in (A), in the presence or absence of 25 μg/mL PF4, in comparison with IgG isolated from vax control serum. VITT ELISA positive n = 5 (patients 2, 3, 4, 6, 12), VITT ELISA negative n = 5 (patients 1, 3, 4, 5, 6), vax controls n = 5. One-way ANOVA with Dunn post hoc test was used for comparison.

The endothelial thromboinflammatory response in VITT is antibody dependent....

Open Access
in Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia > Blood Advances
Published: 2025
Figure 5. The endothelial thromboinflammatory response in VITT is antibody dependent. (A) Platelet fluorescence area per field, neutrophil count per field, and fibrin fluorescence area per field following a 15-minute blood perfusion in the Endo-chip after treatment with VITT ELISA positive serum... More about this image found in The endothelial thromboinflammatory response in VITT is antibody dependent....
Images
Treatment with VITT IgG and PF4 induces endothelial activation. (A) Relative mRNA expression of endothelial cells to glyceraldehyde-3-phosphate dehydrogenase in endothelial cells incubated with media (untreated), treated with VITT IgG and 25 μg/mL PF4, or with vax control IgG and 25 μg/mL PF4. Expression was determined for E-selectin (SELE), P-selectin (SELP), VCAM-1 (VCAM1), ICAM-1 (ICAM1), and VWF. There were n = 3 repeats of n = 3 VITT ELISA positive IgG (patients 3, 4, 11) and n = 3 vax control IgG (patients 1, 2, 5). One-way ANOVA with Dunn post hoc analysis was used for comparisons. (B) Left: release of VWF after stimulation of endothelial cells with thrombin, VITT IgG and 25 μg/mL PF4, vax control IgG and 25 μg/mL PF4, or untreated. Representative images. Right: quantification of the average number of VWF strings per field after stimulation with thrombin, VITT IgG + PF4, vax control IgG + PF4, and untreated. A total of 2 to 3 fields of view were used for n = 3 VITT ELISA positive IgG (patients 3, 4, 11) and n = 3 vax control IgG (patients 1, 2, 5). One-way ANOVA with Tukey post hoc test was used for comparisons. (C) Left: representative immunofluorescent images of TF staining (green) of HUVECs after incubation with IgG isolated from VITT or vax control serum. Nuclei are stained with Hoechst (blue). Right: TF fluorescence intensity units after incubation of HUVECs with VITT IgG or vax control IgG; average of n = 3 fields of view from 4 VITT ELISA positive (patients 2, 4, 6, 13) and 3 vax controls (patients 1, 2, 5). Unpaired t tests were used for comparisons. (D) Left: kinetic curve of Xa generation after treatment of endothelial cells with media (untreated), VITT IgG + 25 μg/mL PF4, vax control IgG + 25 μg/mL PF4, and 10 ng/mL TNF-α (left panel). Right: factor Xa generation after treatment of endothelial cells with VITT IgG + PF4 vs vax control IgG + PF4. There were 3 repeats for 3 VITT ELISA positive IgG samples (patients 4, 6, 13) and 3 vax control IgG samples (patients 1, 2, 5). Unpaired t tests were used for comparison.

Treatment with VITT IgG and PF4 induces endothelial activation. (A) Relati...

Open Access
in Endothelial cell activation enhances thromboinflammation in vaccine-induced immune thrombotic thrombocytopenia > Blood Advances
Published: 2025
Figure 6. Treatment with VITT IgG and PF4 induces endothelial activation. (A) Relative mRNA expression of endothelial cells to glyceraldehyde-3-phosphate dehydrogenase in endothelial cells incubated with media (untreated), treated with VITT IgG and 25 μg/mL PF4, or with vax control IgG and 25 μg... More about this image found in Treatment with VITT IgG and PF4 induces endothelial activation. (A) Relati...