Exosomes/EVs: IMMUNOMODULATORY EFFECTS OF AMNITOIC FLUID EXTRACELLULAR VESICLES IN AN IN-VITRO MODEL OF IMMUNE ACTIVATION

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

Abstracts of the 62nd Annual Scientific Meeting of the British Society for Haematology

The proceedings contain 252 papers. The topics discussed include: immunogenicity of Covid-19 vaccination in patients with myelodysplastic syndromes;antibody responses to SARS-CoV-2 vaccination in patients with acute leukemia and high-risk MDS on active anti-cancer therapies;CD9 derepression drives cellular differentiation and restores immune recognition in pediatric acute myeloid leukemia;follow-up of patients with FLT3-mutated relapsed or refractory acute myeloid leukemia in the phase 3 ADMIRAL trial;efficacy and safety of Maribavir as a rescue treatment for investigator assigned therapy in transplant recipients with refractory or resistant cytomegalovirus infections in the SOLSTICE study: phase 3 trial results;long-term survival benefit of eculizumab treatment in patients with paroxysmal nocturnal hemoglobinuria: data from the international PNH registry;and analysis of anemia persistence and related adverse events in patients with paroxysmal nocturnal hemoglobinuria treated with pegcetacoplan.

The Deglycosylated Form of 1E12, a Monoclonal Anti-PF4 IgG, Strongly Inhibits Antibody-Triggered Cellular Activation in Vaccine-Induced Thrombotic Thrombocytopenia, and Is a Potential New Treatment for Vιττ

[Formula presented] Introduction Vaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in “autoimmune” heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics “autoimmune” HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies. Methods and Results Using a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT. Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed. To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner. Conclusions Our results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. Disclosures: T iele: Bristol Myers Squibb: Honoraria, Other;Pfizer: Honoraria, Other;Bayer: Honoraria;Chugai Pharma: Honoraria, Other;Novo Nordisk: Other;Novartis: Honoraria;Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria;Biomarin/Prosensa: Other, Research Funding;Sagent: Other, Research Funding;Rovi: Other, Research Funding;Gore inc.: Other, Research Funding;Bayer Healthcare: Other, Research Funding;Paringenix: Other, Research Funding;BMS: Honoraria, Other, Research Funding;MSD: Honoraria, Other, Research Funding;Boehringer Ingelheim: Honoraria, Other, Research Funding;Aspen: Honoraria, Other, Research Funding;Portola: Other;Ergomed: Other;Instrument Laboratory: Honoraria;Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.

Flow Cytometric Detection of Procoagulant Properties of Plasma from Patients with Clinically Confirmed Vaccine-Induced Immune Thrombotic Thrombocytopenia

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation;however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4. Methods: Our previously established flow cytometry-based procoagulant platelet assay (using cell death marker GSAO and P-selectin) was modified to incorporate exogenous patient plasma and performed on whole blood from healthy donors screened for FcγRIIa responsiveness (aggregation response to anti-CD9 antibody, ALB6), primed with 5 μM SFLLRN. The assay was performed on Australian patients referred for confirmatory VITT testing with probable VITT (confirmed thrombosis within 4-42 days of ChAdOx1 nCov-19 vaccination, D-Dimer > 5x ULN, platelets < 150 x 10 9/L or falling platelet count) after screening on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Procoagulant response was also measured in presence of 0.5 U/mL and 100 U/mL heparin, monoclonal FcγRIIa blocking antibody, IV.3, and intravenous immunoglobulin, IVIg. Some plasmas were incubated with ChAdOx1 nCoV-19 or SARS-CoV-2 spike protein. Flow cytometry positive patients were also tested by serotonin release assay (SRA) and multiplate aggregometry. Clinical correlation was obtained. Results: Citrated plasma from 49 unique patients with suspected VITT are reported. Plasma from ELISA+ve patients with clinical picture consistent with VITT (n=31), significantly increased the procoagulant platelet proportions in healthy donors in presence of 5 μM SFLLRN (p<0.0001, Figure 1A). This increase was not seen with plasma from healthy donors (n=14);or individuals exposed to ChAdOx1 nCov-19 vaccine without VITT: thrombocytopenic thrombosis patients who were ELISA-ve and SRA-ve (n=14);or low-level ELISA+ve patients without thrombocytopenia who were negative by either multiplate or SRA (n=4). The procoagulant platelet response induced by VITT positive plasma was reduced with low dose heparin (0.5 U/mL, p<0.01) except for 3 patients who showed a heparin-enhancing effect (Figure 1B). High dose heparin (100 U/mL, p<0.0001), IV.3 (10 µg/mL, p<0.0001) or IVIg (10 mg/mL, p<0.0001) abolished the procoagulant response (Figures 1C-D). The in vitro effect of IVIg was predictive of the in vivo response to IVIg therapy (Figure 1E). Addition of SARS-CoV-2 spike protein had no effect on the procoagulant platelet response. ChAdOx1 nCov-19 had an inconsistent effect on procoagulant platelet formation in presence of VITT plasma. Use of donors without a robust aggregation response to ALB6 resulted in false negative results. Conclusion: Induction of FcγRIIa dependent procoagulant response by patient plasma, suppressible by high dose heparin and IVIg, is highly indicative of VITT in the correct clinical circumstance. This assay modification of priming donor platelets from known FcγRIIa responsive donors ith a GPCR agonist to potentiate the ITAM signaling from platelet activating immune complexes, results in a sensitive and specific assay. This may represent a functional platform that can be adopted into diagnostic laboratories to identify patients with platelet-activating antibodies and potentially predict treatment responses. [Formula presented] Disclosures: No relevant conflicts of interest to declare.