Dataset Information

Exploring red blood cells as an antigen delivery system to modulate the immune response towards FVIII in hemophilia A

ABSTRACT: Background: The main complication in hemophilia A treatment is the development of inhibitory antibodies against factor (F)VIII. Immune tolerance induction, the gold standard for eradicating anti-FVIII antibodies, is efﬁcient in only 60% to 80% of cases. This underscores the need for more efﬁcient induction of tolerance in patients with hemophilia A with FVIII inhibitors. Objectives: In this study, we explored whether red blood cells (RBCs) can be utilized as antigen delivery system to modulate the immune response against FVIII. Methods: Two promiscuously HLA-DR–presented peptides derived from the A2 and C1 domains of FVIII were fused to the TAT cell-penetrating peptide and incubated with RBCs. Results: Biotinylated TAT-A2 and TAT-C1 peptides were found to interact with RBCs as shown by ﬂow cytometry and imaging ﬂow cytometry. Moreover, macrophages efﬁciently phagocytosed TAT-FVIII peptide–treated RBCs. Using mass spectrometry– based immunopeptidomics we established that TAT-FVIII peptides were presented on major histocompatibility complex class II of macrophages that phagocytosed TAT peptide–pulsed RBCs. Speciﬁcally, the TAT-A2 peptide exhibited efﬁcient processing and presentation on HLA-DR molecules. Importantly, incubation of TAT-C1 peptide– treated RBCs-loaded macrophages with a FVIII-speciﬁc T-cell hybridoma led to a signiﬁcant increase in IL-2 production, suggesting functional presentation of TAT-C1– derived peptides by macrophages. Conclusion: Our ﬁndings indicate that RBCs can serve as effective vehicle for the delivery of FVIII-derived peptides to antigen-presenting cells. The successful display of T-cell epitopes on antigen-presenting cell using ex vivo–loaded RBC may be potentially utilized to modulate pathogenic immune responses such as observed in a subset of patients with hemophilia A.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

DISEASE(S): Autosomal Hemophilia A

SUBMITTER: Arie Hoogendijk

LAB HEAD: A.J. Hoogendijk

PROVIDER: PXD069058 | Pride | 2025-10-02

REPOSITORIES: Pride

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Exploring red blood cells as an antigen delivery system to modulate the immune response towards FVIII in hemophilia A.

Miranda Mariarosaria M Brandsma Eelke E Robben Lotte L Van Dender Helena H van Alphen Floris P J FPJ Fijnvandraat Karin K van den Biggelaar Maartje M Lacroix-Desmazes Sebastien S van Bruggen Robin R Voorberg Jan J

Journal of thrombosis and haemostasis : JTH 20241129 3

<h4>Background</h4>The main complication in hemophilia A treatment is the development of inhibitory antibodies against factor (F)VIII. Immune tolerance induction, the gold standard for eradicating anti-FVIII antibodies, is efficient in only 60% to 80% of cases. This underscores the need for more efficient induction of tolerance in patients with hemophilia A with FVIII inhibitors.<h4>Objectives</h4>In this study, we explored whether red blood cells (RBCs) can be utilized as antigen delivery syste ...[more]

PMID: 39617188

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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

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.

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