ABSTRACT
Background & Aim: Amniotic fluid (AF)-derived EVs are currently under investigation for use as anti-inflammatory therapeutics in COVID-19 and COVID-19 long haulers. The dysregulation of the immune response induced by SARS-COV-2 is a key driver of both acute COVID-19 induced lung injury and long term COVID-19 sequela. There is a clear need to identify therapeutics that suppress excessive inflammation and reduce immune cell exhaustion to improve patient short term and long-term outcomes. Amniotic fluid (AF)- derived extracellular vesicles (EVs) have previously been shown to deliver anti-inflammatory and immune-modulatory signals to diverse cellular targets. We aimed to test if AF-EVs carry immune-suppressive molecules and can suppress T-cell immune activation and exhaustion in vitro. Methods, Results & Conclusion(s): The AF-EV biologic tested was derived from AF collected from consenting donors during planned, fullterm cesarean sections. AF was centrifuged and filtered to remove cellular debris and create a product containing AF-EVs and soluble extracellular components. Fluorescent EXODOT analysis was performed to demonstrate the presence of EV markers CD9, CD81, ALIX, and immune suppressive molecule PD-L1. T-cell activation/exhaustion was induced in vitro by treating human peripheral blood mononuclear cells with activation agent PHA for 3 days with the addition of AF-EVs or saline control. Immune activation/exhaustion was measured by flow cytometry to determine the expression of PD-1 on CD3+ T-cells. The AF-EV biologic was characterized to contain EVs with positive expression of CD9, CD81, ALIX, and PL-L1. T-cell activation/exhaustion was upregulated in response to PHA and was significantly reduced by 8% in AF-EV treated T-cells compared to saline control (77.7% vs 85.7%, respectively P<0.05). These findings demonstrate that AF-EVs do express PD-L1, a surface marker that has previously been demonstrated to contribute to exosome-mediated immunosuppression. Furthermore, we confirmed in vitro that AF-EVs suppress T-cell activation/ exhaustion in the presence of a T-cell activation agent. COVID-19 long haulers have been described to have upregulated and pro-longed immune activation and T-cell exhaustion, marked by an increase in PD1+ T-cells. Therefore, this finding serves as a starting point for the development of a potential mechanism of action that may describe AF-EV's therapeutic effect in COVID-19 long hauler patients.Copyright © 2023 International Society for Cell & Gene Therapy
ABSTRACT
Background & Aim: Extracellular Vesicles (EVs) isolated from single cell sources such as mesenchymal stem cells and biological fluids such as amniotic fluid have previously been found to have various effects on immune cell functions. Zofin is an acellular biologic derived from amniotic fluid and contains naturally occurring EVs. Zofin is currently under clinical investigation for COVID-19 infection and Post Acute Sequale of COVID-19. Therefore, we aimed to investigate the effects of Zofin on the lymphocyte immune response in an in vitro cell culture model. Methods, Results & Conclusion: Zofin was manufactured from fullterm amniotic fluid collected from healthy, consenting mothers during planned cesarean section. The Zofin used in these experiments contained 1.5x1011 particles/ml as quantified by Nanosight NS300. To study the immune response in vitro, PBMCs were stimulated with 5μg/ml Phytohemagglutinin (PHA) for up to 8 days. PBMCs were labeled before culture with cell trace dye to monitor changes in proliferation. Stimulated PBMCs were treated with either serum-free media (vehicle) or different concentrations of Zofin (10% and 20%). 4 days following PHA stimulation, Zofin suppressed PHA-induced T cell proliferation in a dose-dependent manner. 8 days following PHA stimulation there were higher rates of proliferation, Zofin maintained suppressed proliferation at 20% concentrations. In the same cell culture model, treatment with Zofin attenuated a PHA-induced increase in T cell activation as observed by a reduction in expression of activation markers. Overall, this data demonstrates that Zofin has an effect on the immune response of T cells and suggests amniotic fluid derived EVs could be used as an immunotherapeutic tool
ABSTRACT
Background & Aim: Amniotic fluid (AF)-derived extracellular vesicles (EVs) are currently being studied within clinical trials as a novel therapeutic drug for acute and chronic diseases such as COVID-19, osteoarthritis, and chronic obstructive pulmonary disease (COPD). Recently, AF has been characterized to contain harvestable EVs that reduce cytokine expression and protect from high oxygen tissue injury. However, the reproducibility of EV populations derived from AF samples has yet to be fully explored. Here within, we present results of a fluorescent nanoparticle tracking analysis that characterizes the reproducibility of CD81+ and CD133+ populations of EVs. Methods, Results & Conclusion: AF was collected from consenting adults during planned, full term cesarean sections and then processed via filtration and ultracentrifugation to precipitate the nanoparticle population. Prior to fluorescent analysis, a MACSPlex exosome kit (Miltenyi Biotec) was used to identify potential exosome surface markers. Nine independent samples of AF-derived nanoparticles were used for Zetaview analysis. Each pellet was resuspended in saline and stained with CD133-AF488 and CD81-DyLight55 antibodies. Fluorescent nanoparticle tracking analysis was completed using the Zetaview Quatt (Particle Metrix). Subsequent videos of stained and total particles were recorded to determine the percentage of positive stained nanoparticles. Exosome surface marker analysis revealed 10 out of the 37 tested surface markers to be present with the most intense surface markers being CD81 and CD133. Furthermore, detection of CD81+ and CD133+ nanoparticles via Zetaview was found in all samples (n=9). The AF- derived nanoparticles were CD81+ and CD133+ at 73.51 ± 18.35 % and 30.41 ± 14.06 % (Mean ± SD), respectively. A high percentage of the nanoparticles were CD81+ with a smaller yet apparent display of CD133+ nanoparticles throughout the samples. Fluorescent characterization of the AF-derived samples demonstrated the reproducibility of both CD81+ and CD133+ nanoparticles. Studying the nature of these nanoparticle populations helps researchers to characterize samples consistently and could lead to understanding their therapeutic potential as a novel biologic.