Loss of ADAR1-mediated RNA editing disrupts lineage fidelity in human T cell development by inducing interferon response genes Free

T lymphocytes originate from bone marrow hematopoietic stem cells (HSC) that migrate to the thymus for maturation, selection, and subsequent export to the periphery. Although several studies have focused on transcriptional regulation in this tightly controlled process, the involvement of RNA modification is still unexplored.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR1 (Adenosine Deaminase Acting on RNA 1) is an essential post-transcriptional mechanism that prevents aberrant activation of innate immune sensors such as PKR by modifying endogenous double-stranded RNA (dsRNA). Although ADAR1 is known to be critical for hematopoiesis and immune tolerance, its role in human T cell development is not fully understood.

Here, we utilized a 3D artificial thymic organoid (ATO) system seeded with cord blood-derived CD34⁺ hematopoietic stem and progenitor cells (HSPC) to decipher the functional role of ADAR1 in human T-cell development. ADAR1 was knocked down via lentiviral shRNA, and differentiation was evaluated at multiple time points (3–12 weeks) using flow cytometry and single-cell RNA sequencing (scRNA-seq) of human CD45⁺ cells isolated from early-stage (2-week) and mature-stage (10-week) ATOs.

Our results revealed that ADAR1 knockdown resulted in a developmental block at the Thy2–Thy3 transition, with marked reduction in CD1a⁺ T cell precursors and a significant shift away from the canonical T cell differentiation trajectory. Flow cytometry revealed increased expression of CD56 and CD14, consistent with lineage skewing toward innate and myeloid phenotypes. IL-7 titration (500–5000 U/mL) demonstrated a cytokine dose-dependent accumulation of aberrant CD56⁺ populations in ADAR1-deficient HSPCs. Notably, scRNA-seq of CD45⁺ cells from 2-week ATOs identified a transcriptionally distinct progenitor population exclusive to ADAR1 knockdown conditions. These population co-expressed myeloid and lymphoid genes, including CD7, IL7-R, SPI1 and MPO.

At later time points (10–12 weeks), ADAR1-deficient cultures exhibited reduced numbers of CD4⁺CD8⁺ double-positive cells and increased frequencies of highly inflammatory CD14⁺ monocytes and CD56⁺ NK-like cells. These phenotypes were confirmed by 10-week scRNA-seq analysis that demonstrated reduced CD4 and CD8 expression and activation of interferon-stimulated genes (ISGs). Interestingly, our data also revealed that the deficiency in late-stage T-cell development is driven by a combination of progressive loss of T cell precursors and the persistence of the myeloid/lymphoid progenitors in ADAR1-deficient cells.

We demonstrated that ADAR1-mediated RNA editing is critical for maintaining human T cell lineage fidelity during early thymic development. ADAR1 deficiency leads to suppression of T lineage progression, and emergence of a multi-lineage population with abnormal progenitor and myeloid characteristics and activation of interferon response genes. These studies reveal a mechanistic connection between impaired RNA editing, disrupted T cell development, and the potential for immune dysregulation.