Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Experimental layout of the ARDS model: After 4 hours of intratracheal (IT) LPS injection (3 mg/kg, 100 μl), 30 μg of hep-EVs, hep-SPLEVs, or MSC-EVs, or vehicle [phosphate-buffered saline (PBS)] was injected into the tail vein. Samples were collected and analyzed at 20 hours. h, hours; iv, intravenous. ( B ) Counts of total inflammatory cells and neutrophils in BALF [control ( n = 6), LPS only ( n = 7), LPS + hep-EVs ( n = 4), and LPS + MSC-EVs ( n = 3)]. ( C ) Lung Injury scores were calculated in 10 fields in each sample [control ( n = 4), LPS only ( n = 5), LPS + hep-EVs ( n = 6), and LPS + MSC-EVs ( n = 3)]. ( D ) Hematoxylin and eosin (H&E)–stained images of the control or LPS-challenged lung (left) and lung injury score [LPS only ( n = 5), LPS + hep-EVs ( n = 6), LPS + hep-SPLEVs ( n = 8), and LPS + MSC-EVs ( n = 3)] (right) following each treatment. Scale bars, 100 μm. ( E ) Counts of neutrophils in BALF of LPS-challenged mice following indicated treatments [LPS only ( n = 7), LPS + hep-EVs ( n = 4), LPS + hep-SPLEVs ( n = 5), and LPS + MSC-EVs ( n = 3)]. ( F ) Enzyme-linked immunosorbent assay (ELISA) of cytokines (IL-6 and TNFα) in sera of ARDS-induced mice treated with hep-EVs or hep-SPLEVs ( n = 4 for each). ( G ) Localization of hep-EVs and hep-SPLEVs in the lung. Red: F4/80-positive macrophages; green: hep-EVs or hep-SPLEVs prestained with PKH67; blue: DAPI. Scale bar, 20 μm. A tag-free recombinant human sPLA 2 -X was used in (D) to (G) under the condition shown in table S3. * P < 0.033, ** P < 0.002, and *** P < 0.001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Injection, Saline, Control, Staining, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of phospholipids extracted from LPS-challenged mouse lungs with or without 30 μg of sPLA 2 -X, hep-EVs, hep-SPLEVs, or vehicle (PBS) treatment for 20 hours ( n = 3 for each). Colors indicate fold changes in the levels of individual phospholipids [PC (phosphatidylcholine), pPC (plasmalogen-type PC), PE (phosphatidylethanolamine), pPE (plasmalogen-type PE), PS (phosphatidylserine), PA (phosphatidic acid), PG (phosphatidylglycerol), and PI (phosphatidylinositol)] following each treatment relative to those in control. ( B ) LC-MS/MS analysis of free fatty acids in the lung with same treatment as (A). DPA, docosapentaenoic acid. ( C ) LC-MS/MS analysis of PUFA metabolites in the lung. Colors indicate fold changes in the levels of individual metabolites following each treatment relative to those in control. ( D ) Representative LipiORDER images for phase transition of lipids in the lung of LPS-challenged mice with sPLA 2 -X, hep-EVs, or hep-SPLEVs treatment for 20 hours and normal control (NC). Scale bar, 50 μm. ( E and F ) Representative LipiORDER images for phase transition of lipids and immunostaining of surfactant protein C (SFTPC) in primary mouse alveolar cells (E) and A549 cells (F) with or without LPS stimulation for 6 hours in the presence or absence of hep-EVs or hep-SPLEVs (5 μg/ml). The cells were cultured on collagen-coated ϕ18-mm cover glasses and photographed under a confocal microscope. Scale bar, 50 μm. A tag-free recombinant human sPLA 2 -X was used under the condition shown in tables S3 and S4.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Sublimation, Immunostaining, Cell Culture, Microscopy, Recombinant

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Experimental layout of the BLM-induced acute lung injury model: CL or PBS liposomes (PL) were preinjected into mice 24 hours before IT BLM administration, and after 24 hours, PBS or hep-SPLEVs were injected into the tail vein. After additional 24 hours, samples were collected and analyzed. ( B ) Counts of total inflammatory cells in BALF of mouse lungs challenged with BLM for 24 hours in the presence or absence of hep-SPLEVs [PBS ( n = 5), PL + hep-SPLEVs ( n = 4), CL + hep-SPLEVs ( n = 4), and CL ( n = 4)]. ( C ) H&E-stained images of the lungs (left) and lung injury scores (right) following BLM-induced acute lung injury [PBS ( n = 4), PL + hep-SPLEVs ( n = 4), CL + hep-SPLEVs ( n = 3), and CL ( n = 3)]. N-terminally His-tagged sPLA 2 -X bv was used under the condition shown in table S3. ** P < 0.002, and *** P < 0.001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Liposomes, Injection, Staining

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Gene Ontology singular enrichment analysis based on RNA-seq analysis at 24 hours in the lungs of ARDS-challenged mice treated with 30 μg of hep-EVs, hep-SPLEVs, or with PBS. MAPK, mitogen-activated protein kinase. ( B and C ) Quantitative polymerase chain reaction (qPCR) of Srebf1 in mouse lungs at 20 hours (B) and SREBF1 in A549 cells at 6 hours (C) following each treatment ( n = 3 for each). ( D ) Western blotting of SREBP1, AKT, and phosphorylated AKT (pAKT), as well as α-tubulin as a loading control, in A549 cells following each treatment. Arrow indicates cleaved SREBP1. Right panels show the quantified ratio of cleaved SREBP1 to α-tubulin (top) or pAKT to AKT (bottom) ( n = 3 for each). ( E and F ) The quantified ratio of cleaved SREBP1 to β-actin as a loading control ( n = 3 for each) (E) and Western blotting of SREBP1 and β-actin (F) in mouse lungs after treatment with hep-EVs (5 μg/ml), hep-SPLEVs, or with PBS in the presence or absence of LPS. ( G ) LipiORDER analysis of A549 cells following each treatment. Hep-EVs or hep-SPLEVs were used at 5 μg/ml. Representative images for phase transition of lipids (top) and fluorescence intensity (bottom) are shown. ( H ) LipiORDER images for phase transition of lipids (left) and fluorescence intensity (right) in the lungs of WT and Srebf1 –/– mice following each treatment ( n = 3 for each). Hep-EVs or hep-SPLEVs were used at 30 μg per mouse. ( I ) Analysis of representative phospholipid species in lungs of WT and Srebf1 –/– mice following each treatment. Y axis shows relative peak areas of individual lipids in LPS-challenged mice compared to control mice. Nontagged sPLA 2 -X was used in (A), and N-terminally His-tagged sPLA 2 -X bv was used in (B) to (I) under the condition shown in tables S3 and S4. * P < 0.033, ** P < 0.002, and *** P < 0.001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Control, Sublimation, Fluorescence

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Lung injury scores following treatment with LPS in the presence of hep-EVs, Akata-EVs, hep-SPLEVs, Akata-SPLEVs, or vehicle (PBS). ( B ) Comparison of the ratio of phospholipid classes in hep-EVs versus Akata-EVs. ( C ) qNano analysis of PG PL. Histogram of size distribution (left) and average particle diameter (right) of PG PL before and after treatment with sPLA 2 -X (right) is shown. ( D and E ) Counts of total inflammatory cells and neutrophils in BALF [PBS ( n = 4), hep-EVs ( n = 4), hep-SPLEVs ( n = 5), PG PL ( n = 6), and PG-SPLEVs ( n = 6)] (D) and qPCR of Il6 in the lung ( n = 3 for each) (E) following each treatment in the LPS-induced ARDS model. ( F ) LipiORDER images for transition images of lipids (top) and fluorescence intensity (bottom) in A549 cells following each treatment. Hep-EVs or hep-SPLEVs were used at 5 μg/ml. ( G ) Effects of LPG18:1 on SREBP-1 cleavage and AKT phosphorylation in A549 cells, as evaluated by Western blotting of SREBP1, AKT and pAKT, as well as α-tubulin as a loading control. Quantified ratios of cleaved SREBP1 to α-tubulin ( n = 4) or pAKT to AKT ( n = 3) are shown. Values are mean ± SEM in [(A), (C), (D), (E), and (G)]. The results were analyzed by analysis of variance (ANOVA) test and Tukey’s multiple-comparison test [(A), (C), (D), (E), and (G)] or by Student’s t test (C). N-terminally His-tagged sPLA 2 -X bv was used under the condition shown in tables S3 and S4. * P < 0.033, ** P < 0.002, *** P < 0.001, and **** P < 0.0001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Comparison, Fluorescence, Phospho-proteomics, Western Blot, Control

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Survival curves of mice treated with PBS ( n = 9), hep-EVs ( n = 8), or hep-SPLEVs ( n = 8) in the moderate to severe sepsis model. ( B ) qPCR evaluation of splenic Il6 and Tnf expression in mice treated for 10 days with hep-EVs or hep-SPLEVs after cecum puncture. ( C ) Survival curves after treatment with PBS, hep-EVs, or hep-SPLEVs ( n = 9 for each) in the LPS-induced coagulopathy model. ( D ) Serum ALT and AST levels with or without administration of hep-SPLEVs at 48 hours in the CCl 4 -induced hepatitis model ( n = 3 to 4). ( E to G ) Compared effects of hep-SPLEVs ( n = 9) and hep-EVs ( n = 6) as DSS alone ( n = 10) in DSS-challenged mice. Each mice treated 7 days. (E) Representative photos of colon of mice (left). Decrease in colon length (%) after treatments (right). (F) H&E staining of the colon in mice. Black arrowheads show inflammatory cells, white arrowhead shows loss of intestinal structure, double-headed arrow shows thinning, and black arrow shows edema. Scale bar, 100 μm. (G) Inflammatory and DAI scores in mouse colon. ( H ) H&E staining of mouse lungs with or without H3 influenza infection for 7 days in the presence or absence of hep-SPLEVs (left). Lung injury scores in these mice [naïve ( n = 3), influenza ( n = 4), and influenza + hep-SPLEVs ( n = 5)] are shown (right). ( I ) Viral titers as monitored by qPCR of the H3 influenza gene in the lungs in (H) [naïve ( n = 9), influenza ( n = 9), and influenza + hep-SPLEVs ( n = 13)]. N-terminally His-tagged sPLA 2 -X bv was used under the condition shown in table S3. * P < 0.033, ** P < 0.002, *** P < 0.001, and **** P < 0.0001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Expressing, Staining, Infection

Journal: bioRxiv

Article Title: Endogenous CD28 drives CAR T cell responses in multiple myeloma

doi: 10.1101/2024.03.21.586084

Figure Lengend Snippet: (A) Schematic of second-generation retroviral CAR construct used to generate BCMA targeted human CAR T cells. (B) CAR T cell cytotoxic activity during a 24-hr. co-culture with luciferase-tagged MM.1S (left) or U266 (right) myeloma cells ± abatacept. Data are shown as mean ± SD and representative of hBCMABBζ CAR T cells generated from 3 healthy donors. *p<0.05 by two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. (C) Diagram of experimental setup used to evaluate hBCMABBζ CAR T + abatacept therapy in a human MM xenograft model. NSG mice were intravenously inoculated with 1 × 10 6 MM.1S-luc myeloma cells on day −28 and treated with CAR T cells on day 0. Mice received 200 µg abatacept 3x/week beginning the day before infusion and continuing through endpoint. Tumor burden was monitored by IVIS bioluminescent imaging (BLI) 2x/week. (D) Representative bioluminescent images of MM.1S bearing mice on specified days following CAR T cell infusion. (E) Tumor burden expressed as relative photon flux measured by BLI from MM.1S-luc bearing mice treated with hBCMABBζ CAR T or control T cells ± abatacept (200 µg, 3x/week). Each line represents an individual mouse (n = 7 mice per CAR T cell treated group). (F) Kaplan – Meier analysis of survival of hBCMABBζ CAR T or control T cells ± abatacept (200 µg, 3x/week) treated MM.1S-luc bearing mice (n = 8 - 12 mice per CAR T cell treated group). Median survival of hBCMABBζ CAR T treated mice was >100 days post CAR T cell infusion vs. 55 days for hBCMABBζ CAR T + abatacept treated mice. ****p<0.0001 by log-rank Mantel-Cox test. (G) Tumor burden expressed as relative photon flux measured by BLI from MM.1S high tumor burden mice (inoculated on day −35) treated with hBCMABBζ CAR T or control T cells ± abatacept (200 µg, 3x/week). Each line represents an individual mouse (n = 4 mice per CAR T cell treated group). (H) Kaplan – Meier analysis of survival of hBCMABBζ CAR T or control T cells ± abatacept (200 µg, 3x/week) treated MM.1S-luc high tumor burden mice (n = 4 mice per group). Median survival of hBCMABBζ CAR T treated mice was 80 days vs. 45 days for hBCMABBζ CAR T + abatacept treated mice. ***p<0.0001 by log-rank Mantel-Cox test.

Article Snippet: Bioluminescence was measured 2x/week using an IVIS ® Spectrum In Vivo Imaging System (PerkinElmer) to assess tumor burden.

Techniques: Retroviral, Construct, Activity Assay, Co-Culture Assay, Luciferase, Generated, Comparison, Imaging, Control

Journal: bioRxiv

Article Title: Endogenous CD28 drives CAR T cell responses in multiple myeloma

doi: 10.1101/2024.03.21.586084

Figure Lengend Snippet: (A) Schematic depicting the process of manufacturing mouse CD28 knockout (CD28 iKO ) hBCMAmBBmζ CAR T cell. (B) Surface CD28 protein expression on CD28 fl/fl versus CD28 iKO mouse T cells during CAR T cell manufacture. (C) Median fluorescent intensity (MFI) of CD28 measured by flow cytometry at the conclusion of CD28 fl/fl versus CD28 iKO mouse CAR T manufacture. Data shown as mean ± SD from 10+ independent experiments. *p<0.05, ****p<0.0001 by one-way ANOVA. (D) CD28 fl/fl versus CD28 iKO hBCMAmBBmζ CAR T cell cytotoxic activity during 24 hr. co-culture with luciferase-tagged 5TGM1 hBCMA mouse myeloma cells. Cell viability was assessed by luciferase assay. Data are shown as mean ± SD and representative of at least 3 independent experiments. *p<0.05, **p<0.01 by two-way ANOVA with Tukey’s multiple comparison test. (E) Heatmap representation of culture supernatant mouse cytokine concentrations measured by multiplexed Luminex assays at the conclusion of a 24-hr. co-culture of hBCMAmBBmζ CD28 fl/fl or CD28 iKO CAR T cells with 5TGM1 hBCMA myeloma cells. Log 2 transformed cytokine concentrations represent the mean of 4 independent experiments. (F) Diagram of experimental setup used to evaluate CD28 fl/fl versus CD28 iKO hBCMAmBBmζ CAR T cell therapy in a mouse MM xenograft model. RAG2 -/- mice were inoculated intravenously with 2 × 10 6 5TGM1 hBCMA -luc cells on day −14 and treated with CD28 fl/fl or CD28 iKO CAR T cells on day 0. Tumor burden was monitored by IVIS bioluminescent imaging (BLI) 2x/week through endpoint. (G) Tumor burden expressed as relative photon flux measured by BLI from 5TGM1 hBCMA -luc bearing mice treated with CD28 fl/fl or CD28 iKO hBCMAmBBmζ CAR T or control T cells. Each line represents an individual mouse (n = 6 mice per CAR T cell treated group). (H) Kaplan – Meier analysis of survival of CD28 fl/fl or CD28 iKO hBCMAmBBmζ CAR T cell or control T cell treated 5TGM1 hBCMA -luc bearing mice (n = 6 mice per CAR T cell treated group). Median survival of CD28 fl/fl hBCMAmBBmζ CAR T treated mice was 38 days post-CAR T cell infusion vs. 24 days for CD28 iKO hBCMAmBBmζ CAR T treated mice. *p<0.05, **p<0.01, ***p<0.001 by log-rank Mantel-Cox test. (I) Heatmap representation of cytokine levels in the MM BME 7 days following infusion of CD28 fl/fl or CD28 iKO hBCMAmBBmζ CAR T cells into 5TGM1 hBCMA -luc bearing mice. Bilateral hind limbs were harvested, and BM was flushed into 15 µL PBS for multiplexed cytokine analysis. Log 2 transformed cytokine concentrations represent the mean of 3 mice per group.

Article Snippet: Bioluminescence was measured 2x/week using an IVIS ® Spectrum In Vivo Imaging System (PerkinElmer) to assess tumor burden.

Techniques: Knock-Out, Expressing, Flow Cytometry, Activity Assay, Co-Culture Assay, Luciferase, Comparison, Luminex, Transformation Assay, Imaging, Control

Journal: bioRxiv

Article Title: Endogenous CD28 drives CAR T cell responses in multiple myeloma

doi: 10.1101/2024.03.21.586084

Figure Lengend Snippet: (A) Expansion of CD28 fl/fl versus CD28 iKO hBMCAmBBmζ CAR T cells over the course of manufacturing. ns = not significant by two-way ANOVA. (B) Expression of the proliferation marker Ki-67 in CD28 fl/fl versus CD28 iKO hBCMAmBBmζ CAR T cells following 24 hr. stimulation with 5TGM1 target cells as assessed by flow cytometric analysis. ns = not significant by one-way ANOVA. (C) Diagram of experimental setup. 5TGM1 hBCMA bearing RAG2 -/- mice were treated with CD28 fl/fl or CD28 iKO hBCMAmBBmζ CAR T cells (4D and 4E) or MM.1S bearing NSG mice were treated with hBCMABBζ CAR T cells ± 200μg abatacept on days −1, 1, 3, 5, 7 (4F) and euthanized 7 days post adoptive transfer for blood collection and hind limb BM harvest. (D) CAR T cell frequency assessed by flow cytometry in bone marrow (BM, left) and peripheral blood (right) one week after adoptive transfer of CD28 fl/fl or CD28 iKO hBCMAmBBmζ CAR T cells into 5TGM1 hBCMA myeloma bearing mice. Bars represent mean ± SD, dots indicate individual mice (n = 3-6 mice per group). **p<0.01, ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test. (E) CD28 surface protein expression on BM-infiltrating CD28 fl/fl or CD28 iKO hBCMAmBBmζ CAR T cells one week after adoptive transfer into 5TGM1 hBCMA myeloma bearing mice. (F) CAR T cell frequency assessed by flow cytometry in bone marrow (BM, left) and peripheral blood (right) one week after adoptive transfer of hBCMABBζ CAR T cells into MM.1S myeloma bearing mice ± abatacept. CAR T population identified by surface staining and analyzed by flow cytometry. Bars represent mean ± SD, dots indicate individual mice (n = 4-5 mice per group). ***p<0.001 by two-way ANOVA with Tukey’s multiple comparison test. (G) Representative IVIS bioluminescence images of T-lux luciferase expressing hBCMAmBBmζ CAR T cells ± abatacept (200μg, 3x/week) on day 7 or day 15 after infusion. (H) Quantification of photon flux by IVIS imaging within the hind limb region of interest (ROI) over a four-week period following T-lux hBCMAmBBmζ CAR T cell infusion ± abatacept (200μg, 3x/week) into 5TGM1 hBCMA myeloma bearing mice. Dots represent individual mice (n = 3 mice per group), lines are best-fit sigmoidal curves, and significance was determined by mixed effects modeling.

Article Snippet: Bioluminescence was measured 2x/week using an IVIS ® Spectrum In Vivo Imaging System (PerkinElmer) to assess tumor burden.

Techniques: Expressing, Marker, Adoptive Transfer Assay, Flow Cytometry, Comparison, Staining, Luciferase, Imaging