Non-activated Cells Research Articles

Novel [18F]FPG-interleukin-2 conjugate for monitoring immune checkpoint therapy with positron emission tomography

18F-interleukin-2 based PET imaging of activated T cells serves as a potential tool for non-invasive response prediction, treatment evaluation, and patient stratification in cancer immune checkpoint therapy. Herein, we report the radiolabelling of interleukin-2 (IL-2) with a novel arginine selective bioconjugation reagent, 4-[18F]fluorophenylglyoxal ([18F]FPG). Good non-decay corrected bioconjugation efficiencies of 29 ± 4 % (n = 5) were obtained for the [18F]FPG-IL-2. [18F]FPG-IL-2 uptake by the phytohemagglutinin-activated Jurkat cells (50.5 ± 1.2 %, n = 3) was significantly higher compared to the non-activated Jurkat cells (12.9 ± 1.1 %, n = 3). The [18F]FPG-IL-2 uptake was blocked by the pre-treatment of activated Jurkat cells with excess native IL-2 (22.3 ± 2.2 %, n = 3). Dynamic PET imaging and ex vivo biodistribution study of [18F]FPG-IL-2 in healthy and CT26 tumour bearing mice demonstrated hepatobiliary and renal clearance with minimal uptake in other organs and CT26 tumours. [18F]FPG-IL-2 PET imaging was applied to non-invasively monitor immune checkpoint therapy in CT26 tumour bearing mice, treated with IgG (control), ⍺PD-1 (monotherapy), and ⍺PD-1+⍺CTLA-4 (combination therapy). Significant uptake was observed in the spleens and tumours of the mice in the combination therapy group, which was associated with increased cytotoxic CD8+ T-cell infiltration and reduced tumour volumes. [18F]FPG-IL-2 based PET imaging has the potential to monitor immune checkpoint therapy.

Human decidua basalis mesenchymal stem/stromal cells enhance anticancer properties of human natural killer cells, in vitro.

Mesenchymal stem cells/stromal cells from the Decidua Basalis of the human placenta (DBMSCs) express wide range of effector molecules that modulate the functions of their target cells. These properties make them potential candidate for use in cellular therapy. In this study, we have investigated the consequences of interaction between DBMSCs and natural killer (NK) cells for both cell types. DBMSCs were cultured with IL-2-activated and resting non-activated NK cells isolated from healthy human peripheral blood and various functional assays were performed including, NK cell proliferation and cytolytic activities. Flow cytometry and microscopic studies were performed to examine the expression of NK cell receptors that mediate these cytolytic activities against DBMSCs. Moreover, the mechanism underlying these effects was also investigated. Our findings revealed that, co-culture of DBMSCs and NK cells resulted in inhibition of proliferation of resting NK cells, while proliferation of IL-2 activated NK cells was increased. Contrarily, treatment of DBMSC's with comparatively high numbers of IL-2 activated NK cells, resulted in their lysis, whereas treatment with low numbers resulted in reduction in their proliferation. Cytolytic activity of NK cells against DBMSCs was mediated by several activating NK cell receptors. In spite of the expression of HLA class I molecules by DBMSCs, they were still lysed by NK cells, excluding their involvement in cytolytic activity. In addition, preconditioning NK cells by DBMSCs, enhanced their ability to suppress tumor cell proliferation and in severe cases resulted in their partial lysis. Lysis and decrease of tumor cell proliferation is associated with increased expression of important molecules involved in anticancer activities. We conclude that DBMSCs exhibit dualfunctions on NK cells that enhance their anticancer therapeutic potential.

Treating activated regulatory T cells with pramipexole protects human dopaminergic neurons from 6-OHDA-induced degeneration.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed, a decrease in Treg number and function has been reported in PD. In this context, pramipexole, a dopaminergic receptor agonist used to treat PD symptoms, has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells. Treg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28-activated and non-activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co-cultured with invitro-differentiated human dopaminergic neurons in a cytotoxicity assay with 6-hydroxydopamine (6-OHDA). The role of interleukin-10 (IL-10) was investigated by co-culturing activated IL-10-producing Treg cells with neurons. To further investigate the effect of treatment on Tregs, gene expression in pramipexole-treated, CD3/CD28-activated Treg cells was determined by Fluidigm analysis. Pramipexole-treated CD3/CD28-activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6-OHDA. Pramipexole-treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL-10 release and the activation of genes associated with regulation and neuroprotection. Anti-CD3/CD28-activated Treg cells protect dopaminergic neurons against 6-OHDA-induced damage. In addition, activated, IL-10-producing, pramipexole-treated Tregs also induced a neuroprotective effect, and the supernatants of these co-cultures promoted axonal growth. Pramipexole-treated, activated Tregs altered their gene expression in a concentration-dependent manner, and enhanced TGFβ-related dopamine receptor regulation and immune-related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD.

Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide. Current studies have demonstrated that tumor-derived extracellular vesicles (EVs) from different cancer cell types modulate the fibroblast microenvironment to contribute to cancer development and progression. Here, we isolated and characterized circulating large EVs (LEVs), small EVs (SEVs) and non-EV entities released in the plasma from wild-type (WT) mice and the APCMin/+ CRC mice model. Our results showed that human colon fibroblasts exposed from APC-EVs, but not from WT-EVs, exhibited the phenotypes of cancer-associated fibroblasts (CAFs) through EV-mediated NF-κB pathway activation. Cytokine array analysis on secreted proteins revealed elevated levels of inflammatory cytokine implicated in cancer growth and metastasis. Finally, non-activated cells co-cultured with supernatant from fibroblasts treated with APC-EVs showed increased mRNA expressions of CAFs markers, the ECM, inflammatory cytokines, as well as the expression of genes controlled by NF-κB. Altogether, our work suggests that EVs and non-EV components from APCMin/+ mice are endowed with pro-tumorigenic activities and promoted inflammation and a CAF-like state by triggering NF-κB signaling in fibroblasts to support CRC growth and progression. These findings provide insight into the interaction between plasma-derived EVs and human cells and can be used to design new CRC diagnosis and prognosis tools.

Alterations in lipid metabolism accompanied by changes in protein and carotenoid content as spectroscopic markers of human T cell activation

This work aims to understand better the mechanism of cellular processes accompanying the activation of human T cells and to develop a novel, fast, label-free approach to identify molecular biomarkers for this process. The standard methodology for confirming the activation state of T cells is based on flow cytometry and using antibodies recognizing activation markers. The method provide high specificity detection but may be susceptible to background staining or non-specific secondary antibody reactions. Here, we evaluated the potential of Raman-based molecular imaging in distinguishing non-activated and activated human T cells. Confocal Raman microscopy was performed on T cells followed by chemometrics to obtain comprehensive molecular information, while Stimulated Raman Scattering imaging was used to quickly provide high-resolution images of selected cellular components of activated and non-activated cells. For the first time, carotenoids, lipids, and proteins were shown to be important biomarkers of T-cell activation. We found that T-cell activation was accompanied by lipid accumulation and loss of carotenoid content. Our findings on the biochemical, morphological, and structural changes associated with activated mature T cells provide insights into the molecular changes that occur during therapeutic manipulation of the immune response. The methodology for identifying activated T cells is based on a novel imaging method and supervised and unsupervised chemometrics. It unambiguously identifies specific and unique molecular changes without the need for staining, fixation, or any other sample preparation.

Betulinic Acid Potentiates Mast Cell Degranulation by Compromising Cell Membrane Integrity and Without Involving Fcεri Receptors

ABSTRACT Mast cells play important role in acquired and natural immunity making these favorable therapeutic targets in various inflammatory diseases. Here we observed that, pentacyclic tri terpenoid betulinic acid (BA) treatment resulted in a significantly high number (9%) of cells positive for Hoechst and negative for annexin-V indicating that BA could interfere with plasma membrane integrity. The degranulation of both activated and non-activated mast cells was enhanced upon treatment with BA. The pre-treatment of BA had remarkable effect on calcium response in activated mast cells which showed increased calcium influx relative compared to untreated cells. The results also showed potentially less migration of BA treated mast cells signifying the possible effect of BA on cell membrane. BA treatment resulted in a significant increase in mRNA levels of IL-13 while as mRNA levels of other target cytokines, IL-6 and TNF-α seem to be not affected. Moreover, there was global Increase in phosphorylation of signaling proteins and no significant change in phosphorylation of FcεRI receptors indicating that the effect of BA was independent of signaling cascade or FcεRI receptor mediated mast cell aggregation. Overall, these results portray BA potentiates mast cell effector functions by compromising the membrane integrity and independent of FcεRI involvement.

Immune Analysis Using Vitreous Optical Coherence Tomography Imaging in Rats with Steroid-Induced Glaucoma.

Glaucoma is a multifactorial pathology involving the immune system. The subclinical immune response plays a homeostatic role in healthy situations, but in pathological situations, it produces imbalances. Optical coherence tomography detects immune cells in the vitreous as hyperreflective opacities and these are subsequently characterised by computational analysis. This study monitors the changes in immunity in the vitreous in two steroid-induced glaucoma (SIG) animal models created with drug delivery systems (microspheres loaded with dexamethasone and dexamethasone/fibronectin), comparing both sexes and healthy controls over six months. SIG eyes tended to present greater intensity and a higher number of vitreous opacities (p < 0.05), with dynamic fluctuations in the percentage of isolated cells (10 µm2), non-activated cells (10-50 µm2), activated cells (50-250 µm2) and cell complexes (>250 µm2). Both SIG models presented an anti-inflammatory profile, with non-activated cells being the largest population in this study. However, smaller opacities (isolated cells) seemed to be the first responder to noxa since they were the most rounded (recruitment), coinciding with peak intraocular pressure increase, and showed the highest mean Intensity (intracellular machinery), even in the contralateral eye, and a major change in orientation (motility). Studying the features of hyperreflective opacities in the vitreous using OCT could be a useful biomarker of glaucoma.

Comparison of supercritical CO2 extraction and pressurized fluid extraction for isolation of alkaloids from Anacardium occidentale with the study of its anti-inflammatory activity

In recent years, there has been a growing interest in the therapeutic potential of natural compounds, particularly of plant origin, owing to their demonstrated anti-inflammatory properties. Among these, Anacardium occidentale, commonly known as cashew, has garnered significant attention due to its reputed health benefits. This study aim to establish a correlation between the bioactive compounds contained in the extracts of Anacardium occidentale and its anti-inflammatory activity. Dried Anacardium occidentale leaves powder was used as the extraction matrix. Extraction techniques are maceration, pressurized fluid extraction (PFE), and supercritical fluid extraction (SFE). The preliminary analysis of extracts was made by LC-MS/MS. The Response Surface Methodology (RSM), Principal Component Analysis (PCA), and heat maps were employed to model the influence of experimental conditions on extraction yield and peak area of specific compounds from the plant. To evaluate anti-inflammatory activity, RAW 264.7 cells were cultured, activated with LPS, and treated with varying concentrations of the plant extracts. Cell proliferation was assessed using the XTT assay. Indeed, Anacardium occidentale extracts contain anacardic acids, cardanols, and cardol, with distinct profiles yielded by SFE and ethanol-based methods. RSM shows that temperature and ethanol, as additives to CO2, significantly affect extraction efficiency in both PFE and SFE. Moreover, this composition with SFE demonstrate higher selectivity for specific group of compounds. The extracts exhibit anti-inflammatory properties without cytotoxicity in macrophages, reducing levels of pro-inflammatory proteins COX-2, COX-1, and TLR4 in activated cells. This suggests their potential as anti-inflammatory agents without adverse effects on cell viability or pro-inflammatory protein levels in non-activated cells. Overall, these findings underscore the promising therapeutic potential of Anacardium occidentale extracts in mitigating inflammation, while also providing crucial insights into optimizing the extraction process for targeted compound isolation. Thus, this makes a good prospect for the development of anti-inflammatory drugs from this plant.

Mechanical and structural properties of rat and human lymphocytes after the exposure of the whole blood to X-rays in vitro

Objective. By the means of atomic force microscopy to determine the changes in the parameters of the structural and mechanical properties of peripheral blood lymphocytes induced by the irradiation of whole blood by X-rays and identifying the possibility of assessing a state and radiation-induced lymphocyte death programs by analyzing a set of such parameters.Materials and methods. Whole blood of rats and humans was irradiated with X-rays (1–100 Gy) in vitro. Lymphocytes were isolated from the blood after a day of storage, placed on glass slides, fixed with glutaraldehyde and dried. The study of structural and mechanical properties was carried out with the help of atomic force microscope Bruker Bioscope Resolve in Peak Force QNM mode in air. For the sets of AFM parameters, which included elastic modulus, adhesion force, cell surface roughness and cell sizes, a k-mean clustering of data was carried out for the studied experimental groups.Results. The X-ray irradiation of the blood caused changes in the structural and mechanical properties of lymphocytes measured by AFM at the nanoscale. Clustering analysis of the sets of AFM parameters revealed clusters with similar structure in each experimental group (humans, 6and 16-month rats). The studied four clusters were associated with cell states and cell death programs: non-activated cells, activated cells with increased stiffness, apoptotic cells with reduced stiffness, and cells dying via programs other than apoptotic ones with increased stiffness. Each cluster (cell type) with a specific set of AFM parameters was represented differently in the blood lymphocyte population, depending on the dose of X-rays.Conclusion. The set of ACM parameters of lymphocytes including elastic modulus, adhesion force, roughness, and cell sizes, can be helpful for automatically determining the state and death program of lymphocytes after the local irradiation of humans with the involvement of peripheral blood (for example, after radio-therapeutic causes).

Discrete Model of Combustion in a Sample Consisting of Activated and Nonactivated Cells Separated by Gas Dispersed Interlayers

Discrete Model of Combustion in a Sample Consisting of Activated and Nonactivated Cells Separated by Gas Dispersed Interlayers

Microglial cell response to experimental periodontal disease

ObjectivesMicroglial activation is critical for modulating the neuroinflammatory process and the pathological progression of neurodegenerative diseases, such as Alzheimer's disease (AD). Microglia are involved in forming barriers around extracellular neuritic plaques and the phagocytosis of β-amyloid peptide (Aβ). In this study, we tested the hypothesis that periodontal disease (PD) as a source of infection alters inflammatory activation and Aβ phagocytosis by the microglial cells.MethodsExperimental PD was induced using ligatures in C57BL/6 mice for 1, 10, 20, and 30 days to assess the progression of PD. Animals without ligatures were used as controls. Maxillary bone loss and local periodontal tissue inflammation associated with the development of PD were confirmed by morphometric bone analysis and cytokine expression, respectively. The frequency and the total number of activated microglia (CD45+ CD11b+ MHCII+) in the brain were analyzed by flow cytometry. Mouse microglial cells (1 × 105) were incubated with heat-inactivated bacterial biofilm isolated from the ligatures retrieved from the teeth or with Klebsiella variicola, a relevant PD-associated bacteria in mice. Expression of pro-inflammatory cytokines, toll-like receptors (TLR), and receptors for phagocytosis was measured by quantitative PCR. The phagocytic capacity of microglia to uptake β-amyloid was analyzed by flow cytometry.ResultsLigature placement caused progressive periodontal disease and bone resorption that was already significant on day 1 post-ligation (p < 0.05) and continued to increase until day 30 (p < 0.0001). The severity of periodontal disease increased the frequency of activated microglia in the brains on day 30 by 36%. In parallel, heat-inactivated PD-associated total bacteria and Klebsiella variicola increased the expression of TNFα, IL-1β, IL-6, TLR2, and TLR9 in microglial cells (1.6-, 83-, 3.2-, 1.5-, 1.5-fold, respectively p < 0.01). Incubation of microglia with Klebsiella variicola increased the Aβ-phagocytosis by 394% and the expression of the phagocytic receptor MSR1 by 33-fold compared to the non-activated cells (p < 0.0001).ConclusionsWe showed that inducing PD in mice results in microglia activation in vivo and that PD-associated bacteria directly promote a pro-inflammatory and phagocytic phenotype in microglia. These results support a direct role of PD-associated pathogens in neuroinflammation.

Real-Time Micromotor Probe for Immune Neutrophil Activation State.

Neutrophil activation is a hallmark of the immune response. Approaches to identify neutrophil activation in real time are necessary but are still lacking. In this study, magnetic Spirulina micromotors are used as label-free probes that exhibit differences in motility under different neutrophil activation states. This is correlated with different secretions into the extracellular environment by activated/non-activated cells and local environmental viscoelasticity. The micromotor platform can bypass non-activated immune cells while being stopped by activated cells. Thus, the micromotors can serve as label-free biomechanical probes of the immune cell state. They can detect the activation state of target immune cells in real time and with single-cell precision, which provides new ideas for the diagnosis and treatment of diseases while deepening understanding of the biomechanics of activated immune cells.

Synthesis of Bispecific Antibody Conjugates Using Functionalized Poly-ADP-ribose Polymers.

Poly-ADP-ribose (PAR) is a natural type of polymer derived from enzymatic reactions catalyzed by cellular poly(ADP-ribose) polymerases (PARPs). Given its notable solubility and biocompatibility, the PAR polymer may function as effective carriers for therapeutics in addition to modulating biomolecular interactions in cells. To explore its therapeutic potential, we herein developed a PAR polymer-based bispecific antibody targeting both human epidermal growth factor receptor 2 (HER2) and T-cell CD3 antigens. This was accomplished by conjugating anti-HER2 and anti-CD3 monoclonal antibodies to azido-functionalized PAR polymers through click chemistry. The generated PAR polymer-anti-HER2/anti-CD3 antibody conjugate could not only bind specifically to both HER2- and CD3-expressing target cells but also display potent cytotoxicity against HER2-positive breast cancer cells in the presence of non-activated human peripheral blood mononuclear cells (PBMCs). Functionalized PAR polymers provide a new strategy for synthesizing bispecific antibodies and may enable generation of PAR polymer-based conjugates with unique pharmacological activities for biomedical applications.

Label-Free Characterization of Macrophage Polarization Using Raman Spectroscopy.

Macrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes.

Effects of Cationic Dendrimers and Their Complexes with microRNAs on Immunocompetent Cells.

Short regulatory oligonucleotides are considered prospective tools for immunotherapy. However, they require an adequate carrier to deliver potential therapeutics into immune cells. Herein, we explore the potential of polycationic dendrimers as carriers for microRNAs in peripheral blood mononuclear cells of healthy donors. As an oligonucleotide cargo, we use a synthetic mimic and an inhibitor of miR-155, an important factor in the development and functioning of immunocompetent cells. Dendrimers bind microRNAs into low-cytotoxic polyelectrolyte complexes that are efficiently uptaken by immunocompetent cells. We have shown these complexes to affect the number of T-regulatory cells, CD14+ and CD19+ cell subpopulations in non-activated mononuclear cells. The treatment affected the expression of HLA-DR on T-cells and PD-1 expression on T- and B-lymphocytes. It also affected the production of IL-4 and IL-10, but not the perforin and granzyme B production. Our findings suggest the potential of dendrimer-mediated microRNA-155 treatment for immunotherapy, though the activity of microRNA-dendrimer constructions on distinct immune cell subsets can be further improved.

Quality over quantity; eosinophil activation status will deepen the insight into eosinophilic diseases

Eosinophil associated diseases have gained much attention recently because of the introduction of specific eosinophil targeted therapies. These diseases range from acute parasitic infections to chronic inflammatory diseases such as eosinophilic asthma. In eosinophilic asthma an increased eosinophil cell count in peripheral blood is the gold standard for determination of the pheno-/endotype and severity of disease. Despite a broad consensus there is concern on validity of this simple measurement, because the eosinophil compartment is far from homogenous. Multiple tissues harbour non-activated cells under homeostatic conditions and other tissues, normally devoid of eosinophils, become infested with these cells under inflammatory conditions. It will, therefore, be clear that eosinophils become differentially (pre)-activated at different tissue sites in homeostatic and inflammatory conditions. This complexity should be investigated in detail as it is 1) far from clear what the long-term side effects are that are caused by application of eosinophil targeted therapies in a “one size fits all” concept and 2) real-world data of eosinophil targeted therapies in asthma shows a broad variety in the treatment response. This review will focus on complex mechanisms of eosinophil activation in vivo to create a better view on the dynamics of the eosinophil compartment in health and disease both to prevent collateral damage caused by aberrant activation of eosinophils ánd to improve effectiveness of eosinophil targeted treatments.

Effect of reactive inflammation in osteoarthritis on extracellular traps formation by circulating neutrophils

The results of recent studies demonstrating the presence of pro-inflammatory mediators in osteoarthritis (OA), as well as known similarity of histological signs of inflammation in rheumatoid arthritis (RA) and OA, suggest an important role of NETosis in immune inflammation in OA patients. Our objective was to assess ability of blood neutrophils from ОA patients to generate NETosis spontaneously and after in vitro induction, and impact of reactive synovitis on the dynamics of NETosis indexes. Thirty-one patients with verified OA and 30 healthy volunteers were included into the study. Circulating neutrophils were isolated with one-step density gradient centrifugation using double layers of Iohexol gradient. Subpopulational profile of isolated neutrophil fractions, their viability, and nonspecific activation were evaluated microscopically using Trypan Blue exclusion test, as well as nitro-blue tetrazolium test. NETs were induced by phorbol-12-myristate13-acetate (PMA). Spontaneous and induced formation of NETs was assessed using fluorescence microscopy. The ОA patients were in clinical remission at the time of inclusion in the study. In 23 OA patients, an exacerbation was diagnosed during the study. The neutrophil fractions showed high purity and a high content of viable nonactivated cells. These parameters were comparable in the study groups. Mean percentage of spontaneous NETs in OA patients in remission was significantly increased comparing to healthy controls. Usage of PMA, as inducing agent was accompanied by a significant increase in ability of neutrophils to form NETs. Transition of OA to exacerbation was characterized by further significant increase in spontaneous and PMA-induced NETs. Spontaneous and induced NETs in OA patients at acute stage of the disease are significantly higher than in OA patients in remission state. The growth rate of spontaneous NET formation is 3.74 times higher than the induced NET formation in OA patients upon exacerbation. Statistically significant increase in the ability of peripheral neutrophils to spontaneous and induced formation of extracellular traps was found, depending on the stage of osteoarthritis. The data obtained suggest an opportunity for participation of circulating neutrophils via NETosis in pathogenesis of immune inflammation in OA.

Dasatinib is a potent enhancer for CAR T cell generation by CD3-targeted lentiviral vectors

CD3-targeted lentiviral vectors (CD3-LVs) mediate selective transduction of human T lymphocytes invitro and invivo while simultaneously activating the targeted cells. Previously, we have demonstrated that CD3-LV leads to downmodulation of the CD3:T cell receptor (TCR) complex. We therefore hypothesized that inhibition of CD3 phosphorylation by Src/Abl tyrosine kinase inhibitors such as dasatinib results in enhancement of gene delivery by Tcell-targeted LVs. Indeed, dasatinib treatment of Tcells prior to incubation with CD3-LV increased reporter gene delivery by 3- to 10-fold. Moreover, the presence of dasatinib enhanced selective transduction into non-activated target cells present in whole blood. When combined with delivery of the CD19-chimeric antigen receptor (CAR) gene, dasatinib increased CAR Tcell numbers by close to 10-fold. Importantly, the short-term exposure of Tcells to dasatinib during vector incubation did not interfere with tumor cell killing by the resulting CAR Tcells and rather came along with less upregulated exhaustion markers and a more naive phenotype. Our data suggest that dasatinib prevents CD3-LV-induced phosphorylation and CD3:TCR intake, thereby increasing the amount of CD3-LV bound to the cell surface. This is the first description of dasatinib as transduction enhancer, an activity particularly relevant for CAR Tcell generation with CD3-LV.

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load.

Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs ("mito-EVs") may increase the recipient BEC ATP levels to a greater extent than naïve MVs. We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs-suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases. The online version contains supplementary material available at 10.1007/s12195-022-00738-8.

33219 Development of an in vitro model to study fibrotic response in skin disorders

Dermal fibroblasts are responsible for excessive extracellular matrix remodeling leading to skin fibrosis. Fibrotic remodeling of the skin is associated with growth factors, including TGF-β1 and PDGF-AB. Nitendanib, a tyrosine-kinase inhibitor, is approved for treatment in pulmonary fibrosis. Human dermal fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, ficoll and ascorbic acid for up to 12 days. Cells were activated with either PDGF-AB [3 nM] or TGF-β1 [1 nM]. Nintedanib [1 nM-10 μM] was added at day 0 or 7 together in PDGF-AB or TGF-β1 treated cells. Media were changed twice a week. Non-activated cells (w/o) were used as control. Type I, III and VI collagen formation (PRO-C1, PRO-C3, PRO-C6) were evaluated by ELISAs. Statistical analysis included 2-way ANOVA and Dunnett’s test. PDGF increased PRO-C3 from day 7, peaking at day 10 (P < .0001) and PRO-C6 from day 3 peaking at day 10 (P = .0006) compared with w/o. TGF-β1 increased PRO-C1 from day 3 peaking at on day 10 compared with w/o (P < .0001), and PRO-C3 from day 10 (P < .0001). Nintedanib (≥100 nM) added on either day 0 or 7 reduced PDGF-AB induced PRO-C3 and PRO-C6 to the levels of w/o throughout the study. Nintedanib (≥100 nM) added on day 0 reduced TGF-β1 induced PRO-C1 levels. Treatment with Nintedanib from day 7 (10 μM) reduced TGF-β1 induced PRO-C1 levels. In this model, TGF-β1 and PDGF-AB induced formation of collagens. Nintedanib blocks PDGF-AB-driven collagen formation, while only the highest concentration of Nintedanib was able to inhibit TGF-β1-driven collagen formation.