P068 | MiR-146b restoration in T-large granular lymphocyte leukemia: molecular insights for a new therapeutic strategy against neutropenia development

Introduction. In T-Large Granular Lymphocyte Leukemia (T-LGLL), characterized by the clonal expansion of cytotoxic LGLs, neutropenia is the main clinical manifestation. Current immunosuppressive therapies have limited efficacy and high toxicity, highlighting the need for targeted approaches. Neutropenia development is reported to be due to high Fas Ligand (FasL) production by LGLs, which triggers neutrophil cell death. We recently observed that the downregulation of miR-146b can be responsible for the high expression of FasL, through STAT3-miR-146b-ELAVL1-FasL axis.This project aimed to investigate the molecular consequences of miR-146b restoration in LGLs from both patients and healthy donors (HD), as the first step to proceed toward developing a potential RNA-based therapy for T-LGLL-related neutropenia.Methods. LGLs were isolated from 6 patients, while CD8+ cytotoxic T lymphocytes (CTLs) were purified from 6 HD. Cells were transfected with either a miR-146b mimic or a negative control (scramble). After 24 hours (h), cell viability and transfection efficiency were assessed, and total RNA was extracted to perform RNA sequencing. Gene expression was quantified using CircComPara. Differentially expressed gene (DEG) analysis was conducted using DESeq2 v1.46.0 and gene set enrichment analysis using clusterProfiler v4.14.6 and enrichplot v1.28.0.Results. MiR-146b uptake post-transfection was confirmed with a cell viability >60%. According to transcriptomic data, the changes between scramble and miRNA-transfected conditions are marked in patient LGLs (2,404 DEGs) and mild in HD (154 DEGs).Our data confirmed that ELAVL1 and FasL are over-expressed in T-LGLL patients compared to HD, and demonstrated that miR-146b restoration was able to successfully down-regulate ELAVL1 expression (p