Dataset Information

MHC class II presentation of FVIII-AnnexinA5 fusion proteins internalized by antigen presenting cells

ABSTRACT: The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) remains the most serious complication in the treatment of hemophilia A. While immune tolerance induction (ITI) is the standard strategy to eliminate these antibodies, it fails in approximately 30% of patients with severe hemophilia A, underscoring the need for innovative approaches to promote FVIII-specific tolerance. To address this challenge, we generated fusion proteins composed of A2, A3-C1-C2 (light chain, LCh), and C2 domains of FVIII linked to Annexin A5 (AnxA5), a protein that binds phosphatidylserine (PS), a hallmark of apoptotic cells. ELISA confirmed high-affinity binding of all fusion proteins to immobilized PS. To model PS exposure in vitro, red blood cells (RBCs) were treated with phorbol 12-myristate 13-acetate (PMA), leading to the release of PS-exposing microvesicles. Flow cytometry showed that FVIII-AnxA5 fusion proteins selectively bound to PS-exposing microvesicles but not to intact RBCs. Using mass spectrometry-based immunopeptidomics, we demonstrated that macrophages pulsed with FVIII-AnxA5 fusion proteins efficiently processed and presented FVIII-derived peptides on HLA-DR molecules. These findings suggest that FVIII-AnxA5 fusion proteins can engage apoptotic cell clearance pathways to facilitate antigen presentation in a potentially tolerogenic context. This strategy may offer a novel means of inducing immune tolerance to FVIII in hemophilia A.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

DISEASE(S): Factor Viii Deficiency

SUBMITTER: Arie Hoogendijk

LAB HEAD: A.J. Hoogendijk

PROVIDER: PXD068424 | Pride | 2025-10-20

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20250218_S10_Donor2_MO_LChAV.raw	Raw
	20250218_S11_Donor2_MO_A2AV.raw	Raw
	20250218_S12_Donor2_MO_C2AV.raw	Raw
	20250218_S1_Donor1_MO.raw	Raw
	20250218_S2_Donor1_MO_LCh.raw	Raw

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MHC class II presentation of FVIII-AnnexinA5 fusion proteins internalized by antigen presenting cells.

Miranda Mariarosaria M Leoni Michela M van der Zwaan Carmen C van Bruggen Robin R Reutelingsperger Chris P M CPM Fijnvandraat Karin K Hoogendijk Arie J AJ van den Biggelaar Maartje M Voorberg Jan J

Frontiers in immunology 20250925

<h4>Introduction</h4>The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) remains the most serious complication in the treatment of hemophilia A. While immune tolerance induction (ITI) is the standard strategy to eliminate these antibodies, it fails in approximately 30% of patients with severe hemophilia A, underscoring the need for innovative approaches to promote FVIII-specific tolerance.<h4>Methods</h4>To address this challenge, we generated fusion p ...[more]

PMID: 41080604

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Project description:Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). In addition to genetic mutations in the gene encoding FVIII (F8), there is evidence that other molecular mechanisms may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them, hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-M-CM- -vis the role of nc-RNAs in the regulation of F8 expression and the development of anti-drug antibodies to FVIII infusions used to treat HA. Whole blood samples from nine patients with hemophilia A and five controls were obtained. Among those patients with hemophilia A, three of them were patients who developed neutralizing-anti-FVIII antibodies (inhibitors) and the others are patients without FVIII inhibitors. RNA was isolated and purified from all samples using the RiboPure Blood Kit (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol with modifications to enhance the enrichment of low molecular weight RNAs. All RNA samples were poly (A)-tailed and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). After labeling, the enzyme linked oligosorbent assays (ELOSA) were performed to confirm that the biotin labeling processes were successful. Biotin-labeled RNA samples (500 ng/sample) were hybridized on GeneChip miRNA 3.0 microarrays (Affymetrix). Microarray data were analyzed to identify ncRNAs that were differentially expressed in hemophilia A groups compared to controls.

Project description:Aims Leveraging the tolerogenic nature of their natural clearance pathway, we aim to exploit red blood cells (RBCs) as an antigen delivery system to achieve persistent exposure to ADAMTS13-derived peptides, promoting antigen-specific attenuation of autoreactive CD4+ T cells and induction of regulatory T cells. Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and potentially fatal autoimmune disease caused by a severe ADAMTS13 functional deficiency mediated by autoantibodies targeting ADAMTS13. Despite high survival rates achieved with current treatments, approximately 40% of patients experience relapses. Ensuring longer-lasting recovery by restoring immune tolerance towards ADAMTS13 remains a significant unmet need. Conclusion(s) Our strategy offers a modular and effective method for targeting antigenic peptides to RBCs for transfusion. Antigen-decorated RBCs are efficiently phagocytosed by macrophages and facilitate the presentation of FINVAPHAR on MHC II molecules, giving macrophages the ability to target ADAMTS13-specific T cells. Based on our results we propose that RBCs loaded with ADAMTS13-derived peptides containing immunodominant T cell epitopes represent a promising strategy for promoting tolerance in patients with iTTP. Methods A fusion peptide comprising the TAT cell-penetrating peptide and an immunodominant ADAMTS13-derived T cell epitope (FINVAPHAR core sequence) was designed. Binding of the fusion peptide to RBCs was monitored by flow cytometry and Imagestream. To examine whether this approach can facilitate antigen-presentation on MHC II molecules, peptide-loaded RBCs were fed to macrophages, followed by isolation of HLA-DR-peptide complexes to study the presented peptide repertoire via mass spectrometry. Results We found that the fusion peptide binds to RBCs in a concentration-dependent manner. Peptide-bound RBCs were efficiently phagocytosed by macrophages. Mass spectrometric analysis revealed that phagocytosis of peptide-loaded RBCs by macrophages results in the presentation of FINVAPHAR-containing sequences of varying lengths on MHCII molecules from HLA-DRB1*11 donors, confirming functional antigen presentation