Abstract Topic: 21. Stem cell transplantation - Experimental
Background: Chronic graft versus host disease (cGvHD) is an important late complication of allogeneic stem cell transplantation. CGVHD is a multisystemic alloimmune disorder, mimicking autoimmune disease, that occurs in 30-70% of transplanted patients. Eyes are affected in >50% of patients, resulting in eye irritation, inflammation and infiltration with immune cells, leading to keratoconjunctivitis sicca. A treatment option in addition to systemic therapy is autologous serum eye drops (ASED), where the patient’s own serum is processed into eye drops through centrifugation and dilution to concentrations of 20-50%. Even though ASED is a well-known therapy, its mechanism of action is poorly understood. Extracellular vesicles (EVs) act as communication molecules in the circulation and have been shown to play a role in transplantation and immunology. They show huge potential as novel therapeutics for GvHD, and as clinically translatable GvHD biomarkers.
Aims: This pilot study aimed to evaluate circulatory extracellular molecular biomarkers in ASED from cGvHD patients, compared to healthy population serums, which may inform on the mechanism of action (MOA).
Methods: EVs were isolated by precipitation from the serum of cGvHD patients (N=6) stored during ASED processing and healthy blood donors (N=6). EV size and concentration were assessed by nanoparticle tracking analysis (NTA). EV microRNA cargo (n=800) was evaluated using NanoString technology. Signature microRNA targets were identified using in silico algorithms.
Results: Isolated EVs demonstrated characteristic cup-shaped morphology by transmission electron microscopy and expressed EV-specific markers Alix and Flotillin by western blot. EVs isolated from ASED serums demonstrated significantly larger modal size (ASED average 140nm vs. Ctl average 109nm; p=0.02), but significantly lower RNA yield (ASED average 185ng vs. Ctl average 335ng; p=0.02) compared to healthy control serum. Assessing EV microRNA cargo, ASED EVs expressed an overall higher number of microRNAs than healthy controls (ASED average 91 vs. Ctl average 50 microRNA; p=0.01). There were 228 microRNAs expressed above the lower limit of detection (LOD) in at least 1/12 samples, while 98 microRNAs were expressed in >3 samples per analysis group. Unsupervised hierarchical clustering showed that ASED EVs clustered distinctly from healthy control EVs. There was a signature of 62 microRNAs that were significantly differentially expressed (DE) in ASED EV compared to controls after FDR correction, of which 33 were upregulated and 29 were downregulated in ASED EVs (FC range -6.04-3.35, p-value range p=<0.001-0.047). MIENTURNET target enrichment, focusing on the top 20 most DE microRNAs, identified 48 genes (TargetScan) associated with 15 microRNAs. Interestingly, these mapped to KEGG (TGF-B signaling; miR-106a-5p/miR-17-5p/miR-20a-5p/miR-20b-5p/miR-302d-5p) and REACTOME (retinoid cycle, diseases of visual transduction; miR-106a-5p/miR-17-5p/miR-20a-5p/miR-20b-5p) pathways aligned to vision or ocular GvHD therapy associated MOA. MiRWalk identified 14 microRNAs to target 374 genes and 58 REACTOME pathways when filtered to 3 algorithms (TargetScan, miRDB and miRTarBase), including the TGF-B signaling pathway. Finally, DIANA Tools identified 85 KEGG pathways associated with all 20 highly DE microRNAs, including 53 genes of the TGF-B signaling pathway, mapping to all 20 microRNAs.
Summary/Conclusion: This preliminary study indicates that EVs in the ASED of patients with ocular GvHD may be in part responsible for the therapeutic mechanism of action. EVs show potential for further investigation as a potent ocular GvHD therapy.
Keywords: Graft-versus-host disease (GVHD), HSCT, Cellular therapy